Method and device for purification of blood from circulating cell free dna

ABSTRACT

The invention provides apheresis devices and their use for removal of substantially all types of cell free DNA (cfDNA) in patients&#39; blood, including nucleosome-bound cfDNA, exosome-bound cfDNA and unbound cfDNA (including double stranded DNA (dsDNA), single stranded DNA (ssDNA) and oligonucleotides), to limit the negative effects of the circulating cfDNA and to treat various diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/559,822, filed on Sep. 18, 2017, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The invention provides apheresis devices and their use for removal ofsubstantially all types of cell free DNA (cfDNA) in patients' blood,including nucleosome-bound cfDNA, exosome-bound cfDNA and unbound cfDNA(including double stranded DNA (dsDNA), single stranded DNA (ssDNA) andoligonucleotides), to limit the negative effects of the circulatingcfDNA and to treat various diseases.

BACKGROUND OF THE INVENTION

Circulating extracellular DNA (eDNA), also called cell free DNA (cfDNA),is present in small amounts in the blood of healthy individuals.

Increased levels of circulating cfDNA is now a widely accepted as markerfor a number of diseases and pathological conditions including but notlimited to cancer, metastatic cancer, acute organ failure, organ infarct(including myocardial infarction and ischemic stroke), hemorrhagicstroke, autoimmune disorders, graft-versus-host-disease (GVHD), graftrejection, sepsis, systemic inflammatory response syndrome (SIRS),multiple organ dysfunction syndrome (MODS), graft-versus-host-disease(GVHD), traumatic injury, proinflammatory status in aged individuals,diabetes, atherosclerosis, neurodegenerative disease, autoimmunedisease, eclampsia, infertility, coagulation disorder,pregnancy-associated complications and infection. Different subtypes ofcirculating cell free DNA might play a significant role in progressionof certain diseases and pathological conditions.

It was proposed to use systemic administration of Deoxyribonuclease(DNase) enzyme, which specifically hydrolyzed circulating cfDNA fortreatment of infertility (U.S. Pat. No. 8,916,151); cardiovasculardisorders (U.S. Pat. No. 9,642,822); cancer; sepsis,graft-versus-host-disease (GBHD); organ failure; diabetes;atherosclerosis; delayed-type hypersensitivity reactions (U.S. Pat. Nos.9,248,166; 8,535,663; 7,612,032; 8,388,951; 8,431,123).

However, contrary to early stage animal models, data in real clinicalsettings has shown that systemic application of deoxyribonuclease(DNase) enzyme has limited effects on reducing the quantity ofcirculating cfDNA.

Hazout, A. (PCT/IB2013/056321) has described 10 women with high levelsof circulating cfDNA (>80 ng/μl) treated with 0.1 mg/kg of DNaseI dailyvia intramuscular route twice a day for seven days and observed only anaverage 26% decrease in the level of circulating cfDNA. Theirobservations were in line with Davis et al., who failed to demonstratethe reduction of circulating level of alpha DS DNA in lupus nephritispatients receiving a 25 μg/kg dose of human recombinantdeoxyribonuclease as a total of one intravenous and ten subcutaneousinjections over a period of 19 days despite achievement in plasma ofcatalytically effective deoxyribonuclease concentrations between 40-100ng/ml. (Davis J. C. et al., Recombinant human Dnase I (rhDNase) inpatients with lupus nephritis Lupus (1999) Vol 8 (1), pp. 68-76.)

The most abundant type of circulating cfDNA is represented bynucleosome-bound DNA. A nucleosome is a subunit of nuclear chromatin andconsists of a central core protein formed by an octamer of thedouble-represented core histones and about 147 base pairs ofdouble-stranded DNA (Oudet P, Gross-Bellard M, Chambon P. Electronmicroscopic and biochemical evidence that chromatin structure is arepeating unit. Cell. 1975; 4:281-300). Nucleosome-bound cfDNA mightcirculate in blood as mononucleosomes or higher order structures such asoligonucleosomes or even fragments of chromatin containing over50-100×10³ base pairs of DNA. This particular type of circulating cfDNAoriginates from cells undergoing necrosis or apoptosis. Another sourcecirculating cfDNA is neutrophil NETosis. Neutrophil extracellular traps(NETs), which are extracellular strands of decondensed DNA expelled fromactivated neutrophils, have over 15×10³ base pairs of DNA length thatare organized in 3D net structures of 10-30 nm. NETosis originatingcfDNA might be either particle free or particle bound. NETs also containhighly cytotoxic enzymes and cytrotoxic proteins originating fromneutrophil interior space. (Sørensen, O. E. and Borregaard, N.,Neutrophil extracellular traps—the dark side of neutrophils. J. Clin.Invest. 2016 May 2; 126(5): 1612-20.) It has been shown recently thatnot only neutrophils but also macrophages might produce NET likestructures (Nat Med., 2018, 24(2):232-238).

Another important type of circulating particle bound cfDNA isexosome-bound DNA. Exosomes are small membrane vesicles (30-100 nm) ofexocytotic origin secreted by most cell types that might containsingle-stranded DNA (ssDNA), mitochondrial DNA (mtDNA) anddouble-stranded (dsDNA) of 2.5-10×10³ base pairs at the inner or outerspace of exosome. (Thakur, B. K. et al., Double-stranded DNA inexosomes: a novel biomarker in cancer detection, Cell Research (2014)24:766-769.)

A significant part of circulating cfDNA free of particles is representedby linear and circular dsDNA and ssDNA secreted by cancer cells,activated immune cells and certain other cell types. This type of cfDNAis generally 250-1000 base pairs length or higher and may be enriched inunique genome sequences. (Kumar, P. et al., Normal and cancerous tissuesrelease extrachromosomal circular DNA (eccDNA) into the circulation,Mol. Cancer. Res., Jun. 20, 2017 DOI: 10.1158/1541-7786.MCR-17-0095.)Another important constituent of circulating cfDNA free of particles ismitochondrial DNA (mtDNA) of different lengths.

Another recently discovered type of particle-free circulating cfDNA isrepresented by ultra short double stranded DNA (dsDNA) oligonucleotidesand single stranded DNA (ssDNA) oligonucleotides with a subnucleosomallength (i.e. usually less than ˜147 base pairs). It was shown that thisparticular cfDNA is enriched in mitochondrial DNA (mtDNA), DNA ofmicrobial origin and mutated human genome sequences. (Burnham P.,Single-stranded DNA library preparation uncovers the origin anddiversity of ultrashort cell-free DNA in plasma, Scientific Reports 6,Article number: 27859 (2016), doi:10.1038/srep27859). Importantly, thistype of circulating cfDNA also contains the low molecular weight DNAfragments which are similar of those that appear following degradationof particle bound DNA by DNase I enzyme in blood of patients.

Several attempts have been made to use extracorporeal removaltechnologies to purify patient blood from certain constituents ofcirculating cfDNA pool. See, e.g., U.S. Pat. No. 9,364,601; U.S. PatentApplication Publication No. 2007/0092509; Kusaoi et al., Ther. Apher.Dial, 2016, 20:348-353.

There is a need for new extracorporeal methods of treating diseasesassociated with high circulating level of blood cfDNA and for new moreeffective devices to realize such methods.

SUMMARY OF THE INVENTION

As specified in the Background section, above, there is a need for newextracorporeal methods of treating diseases associated with high levelof circulating blood cfDNA and for new more effective devices to realizesuch methods. The present invention addresses this and other needs byproviding apheresis devices and associated processes.

In one aspect, the invention provides a device configured to performapheresis comprising one or more affinity matrices, wherein said one ormore affinity matrices are capable of capturing nucleosome-bound cellfree DNA (cfDNA), exosome-bound cfDNA, and unbound cfDNA from blood orplasma of a subject.

In some embodiments, the unbound cfDNA comprises dsDNA, ssDNA andoligonucleotides.

In some embodiments, the device of the invention comprises two or moreaffinity matrices. In some embodiments (i) the first one or moreaffinity matrices is capable of capturing nucleosome-bound cell free DNA(cfDNA) and/or exosome-bound cfDNA and (ii) the second one or moreaffinity matrices is capable of capturing unbound cfDNA, and wherein thefirst and second affinity matrices are arranged within the device in anyorder. In some embodiments, (i) the first one or more affinity matricescomprises a DNA binding protein (e.g., a histone [e.g., a H1 histone]),an anti-histone antibody (e.g., an anti-histone H2A antibody), ananti-nucleosome antibody (e.g., AN-1, AN-44), a DNA intercalating agent(e.g., a Hoechst dye such as, e.g., Hoechst 33342), a DNA-bindingpolymer (e.g., a cationic/basic polymer [e.g., polyethylenimine,poly-L-lysine, poly-L-arginine, hexadimethrine bromide, amino terminated(—NH₂) polyamidoamine (PAMAM) dendrimer, polypropyleneimine (PPI)dendrimer], a non-ionic/neutral polymer [e.g., polyvinylpyrrolidone(PVP), polyvinylpolypyrrolidone (PVPP), poly (4-vinylpyridine-N-oxide)],an anionic/acidic polymer; a linear polymer [e.g., polyethylenimine,poly-L-lysine, poly-L-arginine], a branched polymer [e.g.,hyper-branched poly-L-lysine, hyper-branched polyethylenimine], adendrimeric polymer [e.g., polyamidoamine (PAMAM) dendrimer,polypropyleneimine (PPI) dendrimer]), an anti-DNA antibody (e.g., mousemonoclonal IgM Anti-ds+ss DNA antibody ([49/4A1], ab35576, Abeam), alectin (e.g., Galanthus nivalis Lectin (GNA), Narcissus PseudonarcissusLectin (NPA), Conconavalin A, phytohemagluttanin, or cyanovirin), andany combination thereof, and (ii) the second one or more affinitymatrices comprises a DNA binding protein (e.g., a histone [e.g., a H1histone]), a DNA intercalating agent (e.g., a Hoechst dye such as, e.g.,Hoechst 33342), a DNA binding polymer (e.g., a cationic/basic polymer[e.g., polyethylenimine, poly-L-lysine, poly-L-arginine, hexadimethrinebromide, polyamidoamine (PAMAM) amino terminated (—NH₂) dendrimer,polypropyleneimine (PPI) dendrimer], a non-ionic/neutral polymer [e.g.,polyvinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP), poly(4-vinylpyridine-N-oxide)], anionic/acidic polymers; linear polymers[e.g., polyethylenimine, poly-L-lysine, poly-L-arginine], a branchedpolymer [e.g., hyper-branched poly-L-lysine, hyper-branchedpolyethylenimine], a dendrimeric polymer [e.g., polyamidoamine (PAMAM)dendrimer, polypropyleneimine (PPI) dendrimer]), an anti-DNA antibody(e.g., mouse monoclonal IgM Anti-ds+ss DNA antibody ([4914A1], ab35576,Abcam), and any combination thereof. In some embodiments, said two ormore affinity matrices are sequentially arranged as two or more affinitycolumns. In some embodiments, the first affinity matrix in the sequencecomprises a DNA binding polymer (e.g., amino terminated (—NH₂)polyamidoamine (PAMAM) dendrimer, polypropyleneimine (PPI) dendrimer,hyper-branched poly-L-lysine, or hyper-branched polyethylenimine) or aDNA intercalating agent (e.g., Hoechst 33342). In certain embodiments,the affinity matrix is not polyamidoamine (PAMAM) dendrimer.

Non-limiting examples of useful column combinations (arranged in anyorder) are as follows: (a) (i) DNA intercalating agent Hoechst 33342affinity column and (ii) anti-DNA antibody affinity column; or (b) (i)anti-nucleosome antibody affinity matrix (ANAM) column and (ii) anti-DNAantibody affinity column; or (c) (i) anti-nucleosome antibody affinitymatrix (ANAM) column and (ii) polyamidoamine dendrimer affinity matrix(PDAM) column; or (d) (i) anti-nucleosome antibody affinity matrix(ANAM) column and (ii) hyper-branched poly-L-lysine affinity matrix(PLLAM) column; or (e) (i) anti-histone H2A antibody affinity column,(ii) lectin affinity column, and (iii) histone H1 affinity column orpolyamidoamine dendrimer affinity matrix (PDAM) column or hyper-branchedpoly-L-lysine affinity matrix (PLLAM) column or DNA intercalating agentHoechst 33342 affinity column.

In some embodiments, the device of the invention comprises a singleaffinity matrix. Non-limiting examples of useful matrices which can beused as a single affinity matrix include: affinity matrices comprising ahistone (e.g., histone H1 such as, e.g., histone H1.3), affinitymatrices comprising a DNA binding polymer (e.g., a cationic polymer suchas, e.g., amino terminated (—NH₂) polyamidoamine (PAMAM) dendrimer orhyper-branched poly-L-lysine), affinity matrices comprising a DNAintercalating agent (e.g., Hoechst 33342), affinity matrices comprisingan anti-DNA antibody (e.g., mouse monoclonal IgM Anti-ds+ss DNA antibody([49/4A1], ab35576, Abcam). In certain embodiments, the affinity matrixis not polyamidoamine (PAMAM) dendrimer.

In some embodiments, the device of the invention captures at least 30 mgof cfDNA per single apheresis procedure.

In some embodiments, the device of the invention reduces the blood levelof cfDNA by at least 25% per single apheresis procedure. In someembodiments, the device of the invention reduces the blood level ofcfDNA by at least 50% per single apheresis procedure. In someembodiments, the device of the invention reduces the blood level ofcfDNA by at least 75% per single apheresis procedure.

In another aspect, the invention provides a method of reducing the levelof cell free DNA (cfDNA) in the blood of a subject, the methodcomprising: (a) performing an apheresis procedure comprising divertingblood or plasma from the subject into an apheresis device of the presentinvention to produce blood or plasma with reduced levels of the cfDNA;and (b) returning the blood or plasma with reduced levels of the cfDNAto the subject, wherein the apheresis procedure reduces the level ofnucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA in theblood of the subject. In some embodiments, the subject has a diseasecharacterized by elevated level of cfDNA in the blood. In someembodiments, the subject has a disease selected from the groupconsisting of a neurodegenerative disease, a cancer, achemotherapy-related toxicity, an irradiation induced toxicity (e.g.,acute radiation syndrome), an organ failure, an organ injury, an organinfarct, ischemia, an acute vascular event, a stroke,graft-versus-host-disease (GVHD), graft rejection, sepsis, systemicinflammatory response syndrome (SIRS), multiple organ dysfunctionsyndrome (MODS), a traumatic injury, aging, diabetes, atherosclerosis,an autoimmune disorder, eclampsia, infertility, a pregnancy-associatedcomplication, a coagulation disorder, and an infection.

In a further aspect, the invention provides a method of treating adisease in a subject in need thereof, the method comprising: (a)performing an apheresis procedure comprising diverting blood or plasmafrom the subject into an apheresis device of the present invention toproduce the blood or plasma with reduced levels of the cfDNA; and (b)returning the blood or plasma with reduced levels of the cfDNA to thesubject, wherein the apheresis procedure reduces the level ofnucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA in theblood of the subject. In some embodiments, the subject has a diseasecharacterized by elevated level of cfDNA in the blood. Non-limitingexamples of diseases treatable by the methods of the invention include,e.g., a neurodegenerative disease, a cancer, a chemotherapy-relatedtoxicity, an irradiation induced toxicity (e.g., acute radiationsyndrome), an organ failure, an organ injury, an organ infarct,ischemia, an acute vascular event, a stroke, graft-versus-host-disease(GVHD), graft rejection, sepsis, systemic inflammatory response syndrome(SIRS), multiple organ dysfunction syndrome (MODS), a traumatic injury,aging, diabetes, atherosclerosis, an autoimmune disorder, eclampsia,infertility, a pregnancy-associated complication, a coagulationdisorder, and an infection.

In some embodiments of any of the above methods of the invention, themethod further comprises monitoring the level of cfDNA in the blood ofthe subject.

In some embodiments of any of the above methods of the invention, themethod comprises continuing or repeating the apheresis procedure untilthe level of cfDNA is reduced by at least 25%. In some embodiments ofany of the above methods of the invention, the method comprisescontinuing or repeating the apheresis procedure until the level of cfDNAis reduced by at least 50%. In some embodiments of any of the abovemethods of the invention, the method comprises continuing or repeatingthe apheresis procedure until the level of cfDNA is reduced by at least75%.

In some embodiments of any of the above methods of the invention, themethod comprises continuing or repeating the apheresis procedure untilat least 30 mg of cfDNA is removed from the blood of the subject.

In some embodiments of any of the above methods of the invention, theapheresis procedure is repeated two or more times.

In some embodiments of any of the above methods of the invention, theblood for the apheresis procedure is sourced from the portal vein.

In some embodiments of any of the above methods of the invention, theunbound cfDNA comprises dsDNA, ssDNA and oligonucleotides.

In some embodiments of any of the above methods of the invention, thesubject is human.

These and other aspects of the present invention will be apparent tothose of ordinary skill in the art in the following description, claimsand drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an electrophoretic profile of circulating cfDNA from plasmaof a metastatic cancer patient.

FIGS. 2A and 2B show tumors excised from mice treated with DNA accordingto Example 3, where blood was purified with an affinity matrix withanti-histone antibodies and an affinity matrix with lectin fromGalanthus nivalis (snowdrop). FIG. 2A shows tumors excised from controlgroup mice. FIG. 2B shows tumors excised from mice treated with DNA froman NSCLC T3N2M+ patient purified from nucleosome and exosome boundcirculating cfDNA.

FIG. 3 shows an electrophoretic profile of circulating cfDNA from plasmaof a metastatic cancer patient and a stroke patient.

FIG. 4 shows an electrophoretic profile of circulating cfDNA from plasmaof patient with systemic inflammatory response syndrome (SIRS) andmultiple dysfunction syndrome (MODS).

FIG. 5 shows an electrophoretic profile of circulating cfDNA used incell culture experiments.

FIG. 6 shows an electrophoretic profile of circulating cfDNA, DNase Iwestern blot and quantification of DNase I activity and circulatingcfDNA.

FIG. 7 shows an electrophoretic profile of circulating cfDNA from plasmaof a patient with sepsis.

FIG. 8 shows the results of 1% agarose gel electrophoresis of modelplasma enriched with cfDNA prior and following the volume adsorptiontest. Lane 1 is model plasma enriched with cfDNA prior to incubation;lane 2 is model plasma enriched with cfDNA following incubation withethanolamine Sepharose FF control; lane 3 is model plasma enriched withcfDNA following incubation with PDAM; lane 4 is model plasma enrichedwith cfDNA following incubation with PLLAM; lane 5 is model plasmaenriched with cfDNA following incubation with H1.3 affinity matrix.

FIG. 9 shows the results of 1% agarose gel electrophoresis of plasma ofthe patient diagnosed with odontogenic-related sepsis prior to andfollowing the volume adsorption test. Lane 1 is plasma of the patientwith odontogenic-related sepsis following incubation with ethanolamineSepharose FF control; lane 2 is distilled water blank line; lane 3 isplasma of the patient with odontogenic-related sepsis followingincubation with H1.3 affinity matrix; lane 4 is distilled water blankline; lane 5 is plasma of the patient with odontogenic-related sepsisfollowing incubation with PDAM; lane 6 is plasma of the patient withodontogenic-related sepsis following incubation with PLLAM.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

Singular forms “a”, “an”, and “the” include plural references unless thecontext clearly dictates otherwise. Thus, for example, a reference to “amethod” includes one or more methods, and/or steps of the type describedherein and/or which will become apparent to those persons skilled in theart upon reading this disclosure.

The term “about” or “approximately” includes being within astatistically meaningful range of a value. Such a range can be within anorder of magnitude, preferably within 50%, more preferably within 20%,still more preferably within 10%, and even more preferably within 5% ofa given value or range. The allowable variation encompassed by the term“about” or “approximately” depends on the particular system under study,and can be readily appreciated by one of ordinary skill in the art.

The term “device” as used herein refers to any assembly known in the artto enable the purification of liquid solutions, such as, withoutlimitation, e.g., any hollow-ware, a column, a column matrix, a filter,a membrane, a semi-permeable material, a bead (e.g., a microbead or ananobead), or a tubing. The terms “column” and “cartridge” are usedinterchangeably herein in the context of an apheresis device.

The term “affinity matrix” as used herein refers to (i) a solid supportinto which a ligand (e.g., a cfDNA-binding molecule) is immobilized orto (ii) a solid support formed by the ligand itself (e.g., awater-insoluble DNA-binding polymer).

The term “DNA-binding protein” refers to proteins that bind tosingle-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). DNA bindingproteins can bind DNA in sequence-specific manner (e.g., transcriptionfactors and nucleases) or non-sequence specifically (e.g., polymerasesand histones). The linker histone H1 family members are a key componentof chromatin and bind to the nucleosomal core particle around the DNAentry and exit sites.

As used herein, the terms “circulating DNA”, “cell free DNA (cfDNA)”,“circulating cell free DNA (cfDNA)”, “extracellular DNA (eDNA)”, and“circulating extracellular DNA (eDNA)” are used interchangeably to referto DNA present in blood or plasma located outside of circulating cellsof hematopoietic and non-hematopoietic origin.

Nucleosome-bound cfDNA is DNA that is bound to a nucleosome. Anucleosome is a subunit of nuclear chromatin. Nucleosome-bound cfDNAmight circulate in blood as mononucleosomes or higher order structuressuch as oligonucleosomes or even fragments of chromatin containing over50-100×10³ base pairs of DNA. Circulating nucleosome-bound cfDNA mayoriginate from cells undergoing necrosis or apoptosis and fromneutrophil NETosis.

Exosome-bound cfDNA is cfDNA that is bound to exosomes or present inexosomes. Exosomes are small membrane vesicles (about 30-100 nm) ofexocytotic origin secreted by most cell types that might containsingle-stranded DNA (ssDNA), mitochondrial DNA (mtDNA) anddouble-stranded DNA (dsDNA) at the inner or outer space of exosome.

The terms “unbound cfDNA” or “cfDNA free of particles” or “particle freecfDNA” refer to cfDNA which is not hound to exosomes or nucleosomes andencompasses double-stranded DNA (dsDNA), single-stranded DNA (ssDNA),linear or circular and oligonucleotides, including ultrashort DNAmolecules of subnucleosomal size (usually less than 147 base pairs).

As used herein, the terms “subject” and “patient” are usedinterchangeably and refer to animals, including mammals such as humans,veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.),and experimental animal models. In certain embodiments, the subjectrefers to a human patient, including both genders in adult and childpopulations.

In the context of the present invention insofar as it relates to any ofthe disease conditions recited herein, the terms “treat”, “treatment”,and the like mean to relieve or alleviate at least one symptomassociated with such condition, or to slow or reverse the progression ofsuch condition. Within the meaning of the present invention, the term“treat” also denotes to arrest, delay the onset (i.e., the period priorto clinical manifestation of a disease) and/or reduce the risk ofdeveloping or worsening a disease. The terms “treat”, “treatment”, andthe like regarding a state, disorder or condition may also include (1)preventing or delaying the appearance of at least one clinical orsub-clinical symptom of the state, disorder or condition developing in asubject that may be afflicted with or predisposed to the state, disorderor condition but does not yet experience or display clinical orsubclinical symptoms of the state, disorder or condition; or (2)inhibiting the state, disorder or condition, i.e., arresting, reducingor delaying the development of the disease or a relapse thereof (in caseof maintenance treatment) or at least one clinical or sub-clinicalsymptom thereof; or (3) relieving the disease, i.e., causing regressionof the state, disorder or condition or at least one of its clinical orsub-clinical symptoms.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of statistical analysis, molecularbiology (including recombinant techniques), microbiology, cell biology,conjugation chemistry and biochemistry, which are within the skill ofthe art. Such tools and techniques are described in detail in e.g.,Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed.Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.; Ausubelet al. eds. (2005) Current Protocols in Molecular Biology. John Wileyand Sons, Inc.: Hoboken, N.J.; Bonifacino et al. eds. (2005) CurrentProtocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, N.J.;Coligan et al. eds. (2005) Current Protocols in Immunology, John Wileyand Sons, Inc.: Hoboken, N.J.; Coico et al. eds. (2005) CurrentProtocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, N.J.;Coligan et al. eds. (2005) Current Protocols in Protein Science, JohnWiley and Sons, Inc.: Hoboken, N.J.; and Enna et al. eds. (2005) CurrentProtocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, N.J.Hermanson (2013) Bioconjugate Techniques, 3rd ed., Academic Press;Niemeyer (2004) Bioconjugation Protocols: Strategies and Methods,Springer Science & Business Media and Hermanson et al. (1992)Immobilized Affinity Ligand Techniques, Academic Press. Additionaltechniques are explained, e.g., in U.S. Pat. No. 7,912,698 and U.S.Patent Appl. Pub. Nos. 2011/0202322 and 2011/0307437.

Devices and Methods of the Invention

As specified in the Background Section, there is a great need in the artto develop new methods and devices for reducing the level ofsubstantially all types of circulating cfDNA in the blood. The presentdisclosure addresses this and other needs by providing apheresis devicesand methods, wherein the apheresis device reduces the level ofsubstantially all types of of cfDNA, including nucleosome-bound cfDNA,exosome-bound cfDNA and unbound cfDNA (including dsDNA, ssDNA andoligonucleotides).

The use of extracorporeal removal technologies can provide an effectivesolution to eliminate cfDNA from circulation and, correspondingly,decrease the level and negative effects of circulating cfDNA.Therapeutic apheresis is an extracorporeal treatment that removes bloodcomponents from patients; it is used for the treatment of conditions inwhich a pathogenic substance or component in the blood is causingdevelopment of diseases: see for example, Ward M. D., ConventionalApheresis Therapies: A Review Journal of Clinical Apheresis 26:230-238(2011).

Surprisingly, as demonstrated herein, extracorporeal removal ofsubstantially all types of circulating cfDNA has a positive impact onthe treatment of diseases characterized by elevated circulating levelsof cfDNA in the blood.

The present disclosure provides a method for treating diseasescharacterized by elevated circulating levels of cfDNA through theremoval of substantially all types of cfDNA, including nucleosome-boundcfDNA, exosome-bound cfDNA and unbound cfDNA (including dsDNA, ssDNA andoligonucleotides) from the blood of a subject to reduce the negativeeffects of the circulating cfDNA.

Without wishing to be bound by theory, in certain diseases, wherein thelevel of circulating cfDNA is increased, different types of circulatingcfDNA might act in concert by triggering different molecular pathwayseach leading to disease progression and patient mortality; differenttypes of circulating cfDNA acting together might generate synergistictoxicity, i.e.toxic (negative) effect of two or more types ofcirculating cfDNA is greater than the sum of the negative effects ofeach type of cfDNA taken separately.

The inventors have found that removal of substantially all types ofcfDNA, including nucleosome-bound cfDNA, exosome-bound cfDNA and unboundcfDNA (including double stranded DNA [dsDNA], single stranded DNA[ssDNA] and oligonucleotides) from the blood of patients with increasedlevels of circulating cfDNA can effectively reduce or even fully abolishthe pathogenic effects mediated by said circulating cfDNA. Removal ofsubstantially all types of cfDNA, including nucleosome-bound cfDNA,exosome-bound cfDNA and unbound cfDNA (dsDNA, ssDNA andoligonucleotides) appears critical for reducing pathogenic effectsmediated by cfDNA.

The inventors further surprisingly observed that removal ofsubstantially all types of circulating cfDNA might lead to reactivationof endogenous deoxyribonucleases.

It is further described herein that several affinity matrices orcombinations thereof are able to effectively capture substantially alltypes of cfDNA, including nucleosome-bound cfDNA, exosome-bound cfDNAand unbound cfDNA (including dsDNA, ssDNA and oligonucleotides) from theblood of patients in need thereof. Examples of affinity matrices usefulin apheresis devices and methods of the invention include (i) matricescomprising a DNA binding protein (e.g., a histone [e.g., a H1 histone]),(ii) matrices comprising an anti-histone antibody (e.g., an anti-histoneH2A antibody), an anti-nucleosome antibody (e.g., AN-1, AN-44), (iii)matrices comprising a DNA intercalating agent (e.g., a Hoechst dye suchas, e.g., Hoechst 33342), (iv) matrices comprising a DNA-binding polymer(e.g., a cationic/basic polymer [e.g., polyethylenimine, poly-L-lysine,poly-L-arginine, hexadimethrine bromide, amino terminated (—NH₂)polyamidoamine (PAMAM) dendrimer, polypropyleneimine (PPI) dendrimer], anon-ionic/neutral polymer [e.g., polyvinylpyrrolidone (PVP),polyvinylpolypyrrolidone (PVPP), poly (4-vinylpyridine-N-oxide)], ananionic/acidic polymer; a linear polymer [e.g., polyethylenimine,poly-L-lysine, poly-L-arginine], a branched polymer [e.g.,hyper-branched poly-L-lysine, hyper-branched polyethylenimine], adendrimeric polymer [e.g., polyamidoamine (PAMAM) dendrimer,polypropyleneimine (PPI) dendrimer; see, e.g., Kaur et al., J NanopartRes., 2016, 18:146]; see, e.g., U.S. Pat. No. 7,713,701 and Morozov etal., General Reanimatology, 2016, 12:6 for additional examples), (v)matrices comprising an anti-DNA antibody, (vi) matrices comprising alectin (e.g., Galanthus nivalis Lectin (GNA), Narcissus PseudonarcissusLectin (NPA), Conconavalin A, phytohemagluttanin, or cyanovirin), andany combination thereof. In some embodiments, two or more affinitymatrices are sequentially arranged as two or more affinity columns. Insome embodiments, the first affinity matrix in the sequence comprises aDNA binding polymer (e.g., amino terminated (—NH₂) polyamidoamine(PAMAM) dendrimer, polypropyleneimine (PPI) dendrimer, hyper-branchedpoly-L-lysine, or hyper-branched polyethylenimine) or a DNAintercalating agent (e.g., Hoechst 33342).

Described herein are affinity matrices and apheresis devices comprisingsuch matrices. An apheresis device of the invention may be configuredaccording to the knowledge of one of ordinary skill in the art, forexample as described in U.S. Patent Application No. 2017/0035955 (EliazIssac. published Feb. 9, 2017)). In one possible embodiment of theapheresis device, affinity matrices are placed into various affinitycolumns, or cartridges. The apheresis device can comprise a filtrationcartridge and one or more affinity columns having an inlet and anoutlet, in which the device is capable of capturing nucleosome-boundcfDNA, exosome-bound cfDNA and unbound cfDNA (including dsDNA, ssDNA andoligonucleotides), from blood or plasma of a patient. In someembodiments, the device comprises two or more affinity columns insequence. The inlet and outlet can be positioned with respect to theaffinity matrices such that blood entering the inlet must contact theaffinity matrices before exiting through the outlet. Preferably, thegeometry of the device is designed to maximize contact of blood (orplasma) with the affinity matrices during passage through the device. Avariety of such designs are known in the art. For example, the devicecan be a hollow cylinder packed with an affinity ligand immobilized onbeads, having the inlet at one end and the outlet at the opposite end.Other devices, such as microtubule arrays, can be also constructed. Allsuch variations of container geometry and volume and of the affinitymatrices contained therein can be designed according to knownprinciples. In preparing an affinity matrix column, the affinity matrixmay be loaded to at least 50%, 60%, 70%, 75%, 80%, 85%, or 90% columnvolume. A suitable buffer (e.g., PBS, particularly cold PBS) may be usedto equilibrate the column.

In one aspect is provided a histone affinity matrix comprising cellulosebeads and recombinant human histone H1.3, wherein the recombinant humanhistone H1.3 is immobilized on the cellulose beads and wherein the sizeof the beads is between 50 and 350 micrometers. In some embodiments, thesize of the beads is between 100 and 250 micrometers.

In some embodiments, the histone affinity matrix is prepared by aprocess comprising

a) oxidizing cellulose beads having a size between 100 and 250micrometers to yield activated cellulose beads;b) washing the activated cellulose beads;c) preparing a concentrated solution of recombinant human histone H1.3;d) incubating the activated cellulose beads with the concentratedsolution of recombinant human histone H1.3; ande) blocking any free CHO groups on the activated cellulose beads.

In some embodiments, the process further comprises f) washing theactivated cellulose beads with buffer.

In some embodiments, in step a) the cellulose beads are in an aqueoussuspension and oxidized with NaIO. In some embodiments, in step b), theactivated cellulose beads are washed with sodium bicarbonate,hydrochloric acid and water. In some embodiments, step c) comprisesdialyzing a solution of recombinant human histone H1.3 and concentratingthe dialyzed solution in 0.1 M NaHCO₃ at pH 7-9. In some embodiments,the dialyzed solution is concentrated in 0.1 M NaHCO₃ at pH 8. In someembodiments, in step d) the incubation is performed for 3-5 hours at15-30° C. In some embodiments, in step d) the incubation is performedfor 4 hours at room temperature. In some embodiments, in step e) theblocking step comprises adding 1 M ethanolamine to the activatedcellulose beads and reacting for 30 minutes to 2 hours at 15-30° C. Insome embodiments, in step f) the activated cellulose beads are washedwith TBS buffer.

Also provided is a column comprising the histone affinity matrix of anyof the aspects and embodiments above.

In another aspect is provided a lectin affinity matrix preparedaccording to a process comprising

a) reacting lectin with activated agarose beads to yield lectin-coupledagarose; andb) washing the lectin-coupled agarose with buffer.

In some embodiments, the lectin is from Galanthus nivalis (snowdrop). Insome embodiments, the activated agarose beads are CNBr activated agarosebeads. In some embodiments, the buffer is PBS, such as sterile cold PBSat pH 7.2-7.4.

Also provided is a column comprising the lectin affinity matrix of anyof the aspects and embodiments above.

In yet another aspect is provided a polyamidoamine dendrimer affinitymatrix (PDAM) prepared by a process comprising

a) washing cellulose beads with ethanol and water;b) incubating the washed cellulose beads with (±)-epichlorohydrin andNaOH to yield activated cellulose beads;c) reacting the activated cellulose beads with polyamidoamine (PAMAM)dendrimer to yield PDAM beads and removing PAMAM dendrimer that did notreact with the activated cellulose beads; andd) blocking unconverted epoxy groups on the PDAM beads.

In some embodiments, the process further comprises e) washing the PDAMbeads with 0.1 M phosphate buffer and water.

In some embodiments, in step a) the cellulose beads are washed with 98%ethanol and distilled water. In some embodiments, in step b) the washedcellulose beads are incubated with a mixture of (±)-epichlorohydrin and2.5 M NaOH. In some embodiments, in step c), the activated cellulosebeads are suspended with a 20% solution of PAMAM dendrimer with anethylenediamine core. In some embodiments, in step c) the suspending isconducted at 20-30° C. for 3-6 hours. In some embodiments, in step c)the suspending is conducted at 24° C. for 5 hours.

Also provided is a column comprising a PAMAM dendrimer affinity matrix(PDAM) described above. In some embodiments, the column is a PTFE columnand the polyamidoamine dendrimer affinity matrix is sterilized.

In another aspect is provided an anti-DNA antibody affinity matrixprepared by a process comprising

a) preparing activated agarose beads by crosslinkingN-hydroxysuccinimide with agarose beads;b) washing the activated agarose beads with coupling buffer comprisingNaHCO₃ and NaCl;c) adding an antibody against double stranded and single stranded DNA tothe coupling buffer;d) incubating the coupling buffer comprising the antibody with theactivated agarose beads to yield the anti-DNA antibody affinity matrix;ande) washing the anti-DNA antibody affinity matrix with coupling bufferand acetate buffer.

In some embodiments, the agarose beads have a mean size of 90micrometers. In some embodiments, the coupling buffer comprises 0.2 MNaHCO₃ and 0.5 M NaCl and is at pH 8.3. In some embodiments, theantibody is a monoclonal antibody. In some embodiments, the antibody isa mouse antibody. In some embodiments, the washing step is performed atleast three times. In some embodiments, the acetate buffer is 0.1 Macetate buffer at pH 4.0.

Also provided is a column comprising an anti-DNA antibody affinitymatrix described above.

In some embodiments, the column is prepared by incubating the anti-DNAantibody affinity matrix with sterile Tris-HCl buffer. In someembodiments, the sterile Tris-HCl buffer is at pH 7.4.

In another aspect is provided an anti-nucleosome antibody affinitymatrix (ANAM) prepared by a process comprising

a) preparing activated agarose beads by crosslinkingN-hydroxysuccinimide with agarose beads;b) washing the activated agarose beads with coupling buffer comprisingNaHCO₃ and NaCl;c) adding to the coupling buffer an antibody that binds to nucleosomes,wherein the antibody is prepared in a MRL/Mp (−)+/+mouse;d) incubating the coupling buffer comprising the antibody with theactivated agarose beads to yield the anti-nucleosome antibody affinitymatrix; ande) washing the anti-nucleosome antibody affinity matrix with couplingbuffer and acetate buffer.

In some embodiments, the matrix binds to nucleosome bound circulatingcfDNA, and the matrix does not bind to unbound cfDNA that includesdsDNA, ssDNA and oligonucleotides.

Also provided is a column comprising an anti-nucleosome antibodyaffinity matrix (ANAM) described above.

In yet another aspect is provided a DNA intercalating agent Hoechst 3342affinity matrix prepared by a process comprising

a) oxidizing cellulose beads;b) washing the oxidized cellulose beads;c) reacting the washed oxidized cellulose beads with a solutioncomprising Hoechst 33342 and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) to yield Hoechst 33342 immobilized cellulose beads;andd) washing the Hoechst 33342 immobilized cellulose beads.

In some embodiments, in step a) the cellulose beads are oxidized withNaIO for 3-5 hours. In some embodiments, in step b) the oxidizedcellulose beads are washed with 1 M sodium bicarbonate, 0.1 Mhydrochloric acid and water. In some embodiments in step c), thesolution is a pH buffered solution. In some embodiments in step d), thewashing is conducted at least three times.

In another aspect is provided a hyper-branched poly-L-lysine affinitymatrix (PLLAM) prepared by a process comprising

a) dissolving L-lysine monohydrochloride in water and neutralizing withKOH to yield an L-lysine solution;b) heating the L-lysine solution to yield a solution comprisinghyper-branched poly-L-lysine;c) removing the L-lysine and salt from the solution comprisinghyper-branched poly-L-lysine;d) fractionating the solution comprising hyper-branched poly-L-lysine toobtain a fraction comprising hyper-branched poly-L-lysine with anaverage molecular weight of 21,000 to 32,000;e) dialyzing and lyophilizing the fraction comprising hyper-branchedpoly-L-lysine with an average molecular weight of 21,000 to 32,000 toyield a lyophilizate;f) dissolving the lyophilizate in distilled water and dialyzing againstNaHCO₃ to yield a solution comprising HBPL; andg) incubating the solution comprising hyper-branched poly-L-lysine withcyanogen bromide-activated Sepharose 4B suspended in NaHCO₃ to preparehyper-branched poly-L-lysine affinity matrix.

In some embodiments, in step b) the L-lysine solution is heated to 150°C. for 48 hours under a stream of nitrogen. In some embodiments, in stepc), the solution comprising hyper-branched poly-L-lysine is dialyzedagainst water. In some embodiments, in step d) the fractionation isconducted with a size exclusion column. In some embodiments, in step d),the fractionation is conducted with a gel filtration column.

Also provided is a column comprising a hyper-branched poly-L-lysineaffinity matrix (PLLAM) described above.

In yet another aspect is provided a device configured to performapheresis comprising one or more affinity columns comprising an affinitymatrix and configured to remove substantially all types of cfDNA fromthe blood or plasma of a patient. In some embodiments, the devicecomprises two or more affinity columns in sequence. In some embodiments,the device further comprises a filtration cartridge. In someembodiments, the filtration cartridge has an inlet and an outlet. Insome embodiments, one or more of the affinity columns has an inlet andan outlet.

In some embodiments, the device comprises two or more of the followingaffinity columns arranged in any sequence:

a) a column comprising a DNA binding protein (e.g., histone) affinitymatrix;b) a column comprising a lectin (e.g., Galanthus nivalis Lectin (GNA),Narcissus Pseudonarcissus Lectin (NPA), Conconavalin A,phytohemagluttanin, or cyanovirin) affinity matrix;c) a column comprising a DNA binding polymer (e.g., a cationic polymersuch as, e.g., amino terminated (—NH₂) PAMAM dendrimer, hyper-branchedpoly-L-lysine or hyper-branched polyethylenimine) affinity matrix;d) a column comprising an anti-DNA antibody affinity matrix;e) a column comprising a DNA intercalating agent (e.g., Hoechst 3342)affinity matrix;f) a column comprising an anti-nucleosome antibody affinity matrix(ANAM); andg) a column comprising an anti-histone antibody affinity matrix.

In some embodiments, the device comprises one of the following columncombinations arranged in any order:

(a) (i) DNA intercalating agent Hoechst 33342 affinity column and (ii)anti-DNA antibody affinity column; or(b) (i) anti-nucleosome antibody affinity matrix (ANAM) column and (ii)anti-DNA antibody affinity column; or(c) (i) anti-nucleosome antibody affinity matrix (ANAM) column and (ii)polyamidoamine dendrimer affinity matrix (PDAM) column; or(d) (i) anti-nucleosome antibody affinity matrix (ANAM) column and (ii)hyper-branched poly-L-lysine affinity matrix (PLLAM) column; or(e) (i) anti-histone H2A antibody affinity column, (ii) lectin affinitycolumn, and (iii) histone H1 affinity column or polyamidoamine dendrimeraffinity matrix (PDAM) column or hyper-branched poly-L-lysine affinitymatrix (PLLAM) column or DNA intercalating agent Hoechst 33342 affinitycolumn.

In another aspect is provided an apheresis device comprising afiltration cartridge and one or more affinity columns having an inletand an outlet, in which the device is capable of capturing substantiallyall types of cfDNA, including nucleosome bound cfDNA, exosome boundcfDNA and unbound cfDNA (including dsDNA, ssDNA and oligonucleotides),from blood or plasma of a patient.

In some embodiments, the device comprises two or more affinity columnsin sequence. In some embodiments, the first affinity column in thesequence comprises a DNA binding polymer or a DNA intercalating agent.

In some embodiments, the device comprises a column comprising a histoneaffinity matrix upstream of, or before, a column comprising a lectinaffinity matrix. In some embodiments, the device comprises a columncomprising the histone affinity matrix upstream of, or before, a columncomprising a lectin affinity matrix upstream of, or before, a columncomprising a PAMAM affinity matrix. In some embodiments, the devicecomprises a column comprising an anti-DNA antibody affinity matrixupstream of, or before, a column comprising a Hoechst 3342 affinitymatrix. In some embodiments, the device comprises a column comprising ananti-nucleosome antibody affinity matrix upstream of, or before, acolumn comprising a PAMAM affinity matrix.

In some embodiments, the apheresis device captures at least 30 mg ofcfDNA per single apheresis procedure. In some embodiments, the affinitycolumn comprises an immobilized moiety effective to capture one or moreof nucleosome-bound cfDNA, exosome-bound cfDNA and unbound cfDNA,including dsDNA, ssDNA and oligonucleotides. In some embodiments, theimmobilized moiety is selected from the group consisting of: DNA bindingantibody, DNA intercalating agent, DNA binding protein, DNA bindingpolymer, lectin, anti-nucleosome antibody, and anti-histone antibody.

In some embodiments, the DNA binding protein is histone H1 (e.g., H1.3).

In some embodiments, the DNA binding polymer is a cationic polymer. Insome embodiments, the cationic polymer is poly-L-lysine. In someembodiments, the poly-L-lysine is hyper-branched poly-L-lysine. In someembodiments, the cationic polymer is polyethylenimine. In someembodiments, the polyethylenimine is hyper-branched polyethylenimine. Insome embodiments, the cationic polymer is amino terminated (—NH₂)polyamidoamine (PAMAM) dendrimer.

In some embodiments of the above, the apheresis device comprises twosequential affinity columns, in which one column captures nucleosomebound DNA and exosome-bound DNA and another column captures unboundcfDNA including dsDNA, ssDNA and oligonucleotides. In some embodiments,the immobilized moiety is selected from the group consisting of acombination of two or more of the following moieties: DNA bindingantibody, DNA intercalating agent, DNA binding protein, DNA bindingpolymer, lectin, anti-nucleosome antibody, or anti-histone antibody.

In another aspect is provided a method of reducing the level of cfDNA inthe blood of a patient. The method comprises (a) performing an apheresisprocedure comprising diverting blood or plasma from the patient into anapheresis device to produce purified blood or plasma with reduced levelsof cfDNA; and (b) returning the purified blood or plasma to the patient.The apheresis procedure reduces the level of substantially all types ofcfDNA in the patient's blood, including nucleosome-bound cfDNA,exosome-bound cfDNA and unbound cfDNA (including dsDNA, ssDNA andoligonucleotides).

In some embodiments, the method is effective to treat one or more ofmultiorgan failure, a neurodegenerative disease (e.g., Alzheimer'sdisease), cancer, sepsis, septic kidney injury, irradiation inducedtoxicity (e.g., acute radiation syndrome), and chemotherapy-relatedtoxicity.

In some embodiments, the patient has a disease selected from the groupconsisting of cancer, metastatic cancer, acute organ failure, organinfarct, hemorrhagic stroke, graft-versus-host-disease (GVHD), graftrejection, sepsis, systemic inflammatory response syndrome (SIRS),multiple organ dysfunction syndrome (MODS), irradiation induced toxicity(e.g., acute radiation syndrome), chemotherapy-related toxicity,traumatic injury, pro-inflammatory status in aged individuals, diabetes,atherosclerosis, neurodegenerative disease, autoimmune disease,eclampsia, infertility, coagulation disorder, and infection.

In some embodiments, the method is effective to treat a disorder in apatient, wherein the disorder is selected from cancer, metastaticcancer, acute organ failure, organ infarct (including myocardialinfarction and ischemic stroke, hemorrhagic stroke, autoimmunedisorders, graft-versus-host-disease (GVHD), graft rejection, sepsis,systemic inflammatory response syndrome (SIRS); multiple organdysfunction syndrome (MODS); graft-versus-host-disease (GVHD), traumaticinjury, proinflammatory status in aged individuals, diabetes,atherosclerosis, neurodegenerative disease, autoimmune disease,eclampsia, infertility, coagulation disorder, pregnancy-associatedcomplications and infection. In some embodiments, the patient is in needof treatment of the disorder.

In yet another aspect is provided a method for treating multiple organdysfunction syndrome (MODS) in a patient. The method comprises (a)performing an apheresis procedure comprising diverting blood or plasmafrom the patient into an apheresis device to produce purified blood orplasma; and (b) returning the purified blood or plasma with reducedlevels of the cfDNA to the patient. The apheresis procedure reduces thelevel of substantially all types of cfDNA in the patient's blood,including nucleosome-bound cfDNA, exosome-bound cfDNA and unbound cfDNA(including dsDNA, ssDNA and oligonucleotides). In some embodiments, thepatient is in need of treatment of MODS.

In another aspect is provided a method for treating a neurodegenerativedisease in a patient. The method comprises (a) performing an apheresisprocedure comprising diverting blood or plasma from the patient into anapheresis device to produce purified blood or plasma with reduced levelsof cfDNA; and (b) returning the purified blood or plasma to the patient.The apheresis procedure reduces the level of substantially all types ofcfDNA in the patient's blood, including nucleosome-bound cfDNA,exosome-bound cfDNA and unbound cfDNA (including dsDNA, ssDNA andoligonucleotides). In some embodiments, the patient is in need oftreatment of the neurodegenerative disease.

In another aspect is provided a method for treating Alzheimer's diseasein a patient. The method comprises (a) performing an apheresis procedurecomprising diverting blood or plasma from the patient into an apheresisdevice to produce purified blood or plasma with reduced levels of cfDNA;and (b) returning the purified blood or plasma to the patient. Theapheresis procedure reduces the level of substantially all types ofcfDNA in the patient's blood, including nucleosome-bound cfDNA,exosome-bound cfDNA and unbound cfDNA (including dsDNA, ssDNA andoligonucleotides). In some embodiments, the patient is in need oftreatment of Alzheimer's disease.

In another aspect is provided a method for treating cancer in a patient.The method comprises (a) performing an apheresis procedure comprisingdiverting blood or plasma from the patient into an apheresis device toproduce purified blood or plasma with reduced levels of cfDNA; and (b)returning the purified blood to the patient. The apheresis procedurereduces the level of substantially all types of cfDNA in the patient'sblood, including nucleosome-bound cfDNA, exosome-bound cfDNA and unboundcfDNA (including dsDNA, ssDNA and oligonucleotides). In someembodiments, the patient is in need of treatment of cancer.

In another aspect is provided a method for treating sepsis in a patient.The method comprises (a) performing an apheresis procedure comprisingdiverting blood or plasma from the patient into an apheresis device toproduce purified blood or plasma with reduced levels of cfDNA; and (b)returning the purified blood or plasma to the patient. The apheresisprocedure reduces the level of substantially all types of cfDNA in thepatient's blood, including nucleosome-bound cfDNA, exosome-bound cfDNAand unbound cfDNA (including dsDNA, ssDNA and oligonucleotides). In someembodiments, the patient is in need of treatment of sepsis.

In another aspect is provided a method for treating a kidney injury in apatient. The method comprises (a) performing an apheresis procedurecomprising diverting blood or plasma from the patient into an apheresisdevice to produce purified blood or plasma with reduced levels of cfDNA;and (b) returning the purified blood or plasma to the patient. Theapheresis procedure reduces the level of substantially all types ofcfDNA in the patient's blood, including nucleosome-bound cfDNA,exosome-bound cfDNA and unbound cfDNA (including dsDNA, ssDNA andoligonucleotides). In some embodiments, the patient is in need oftreatment of the kidney injury.

In another aspect is provided a method for treating chemotherapy-relatedtoxicity in a patient. The method comprises (a) performing an apheresisprocedure comprising diverting blood or plasma from the patient into anapheresis device to produce purified blood or plasma with reduced levelsof cfDNA; and (b) returning the purified blood or plasma to the patient.The apheresis procedure reduces the level of substantially all types ofcfDNA in the patient's blood, including nucleosome-bound cfDNA,exosome-bound cfDNA and unbound cfDNA (including dsDNA, ssDNA andoligonucleotides). In some embodiments, the patient is in need oftreatment of chemotherapy-related toxicity.

In another aspect is provided a method for treating irradiation inducedtoxicity (e.g., acute radiation syndrome) in a patient. The methodcomprises (a) performing an apheresis procedure comprising divertingblood or plasma from the patient into an apheresis device to producepurified blood or plasma with reduced levels of cfDNA; and (b) returningthe purified blood or plasma to the patient. The apheresis procedurereduces the level of substantially all types of cfDNA in the patient'sblood, including nucleosome-bound cfDNA, exosome-bound cfDNA and unboundcfDNA (including dsDNA, ssDNA and oligonucleotides). In someembodiments, the patient is in need of treatment of irradiation inducedtoxicity.

In some embodiments of any of the above methods, the blood is divertedfrom the portal vein of the patient.

In some embodiments of the above, the purified blood has reduced levelsof cfDNA as compared to the levels of cfDNA in the blood from thepatient prior to the apheresis procedure.

In some embodiments, the purified blood has reduced levels of all ofnucleosome-bound cfDNA, exosome-bound cfDNA and unbound cfDNA, includingdsDNA, ssDNA and oligonucleotides. In some embodiments, the methodfurther comprises periodically monitoring the level of the circulatingcfDNA in the patient's blood, and continuing the apheresis procedure toreduce the circulating level of cfDNA by at least 25% before concludingthe apheresis procedure. In some embodiments, the method furthercomprises periodically monitoring the level of the circulating cfDNA inthe patient blood, and continuing the apheresis procedure on the patientto reduce the circulating levels of cfDNA by at least 50% beforeconcluding the apheresis procedure. In some embodiments, the methodfurther comprises periodically monitoring the level of the circulatingcfDNA in the patient blood, and continuing the apheresis procedure onthe patient to reduce the levels of circulating cfDNA by at least 75%before concluding the apheresis procedure.

In some embodiments of any of the above, at least 30 mg of cfDNA isremoved from the blood from the patient during one or several sequentialapheresis procedures.

In some embodiments of the above, the method steps are repeated, orundertaken on a schedule. The method steps may be conducted twice a day,every day, every two days, every three days, every four days, every fivedays, every six days, every week, every eight days, every nine days,every 10 days, every 11 days, every 12 days, etc. Samples of blood maybe taken from the patient and tested for levels of cfDNA to assess thefrequency of conducting the methods of treatment.

Arrangement of affinity columns in sequence can allow capturing ofsubstantially all types of cfDNA, including nucleosome-bound cfDNA,exosome-bound cfDNA and unbound cfDNA (including dsDNA, ssDNA andoligonucleotides), from blood or plasma of a patient.

Various sequences are described herein and any sequence can be used. Insome embodiments, the device comprises a column comprising a histoneaffinity matrix upstream of, or before, a column comprising a lectinaffinity matrix. In some embodiments, the device comprises a columncomprising the histone affinity matrix upstream of, or before, a columncomprising a lectin affinity matrix upstream of, or before, a columncomprising a polyamidoamine dendrimer affinity matrix (PDAM). In someembodiments, the device comprises a column comprising an anti-DNAantibody affinity matrix upstream of, or before, a column comprising aHoechst 3342 affinity matrix. In some embodiments, the device comprisesa column comprising an anti-nucleosome antibody affinity matrix (ANAM)upstream of, or before, a column comprising a polyamidoamine dendrimeraffinity matrix (PDAM).

As part of the various aspects described throughout the application, is(a) performing an apheresis procedure comprising diverting blood orplasma from the patient into an apheresis device to produce purifiedblood or plasma; and (b) returning the purified blood or plasma withreduced levels of the cfDNA to the patient.

The apheresis device may comprise a histone affinity matrix. The histoneaffinity matrix may comprise recombinant human histone H1.3. The histoneaffinity matrix may be part of an affinity column. The beads used assupport in a histone affinity matrix column may be cellulose beads thatare oxidized with an oxidant before coupling with histone. The beads canbe sepharose beads, for example. Alternatively, support of forms besidesbeads can be used (hollow fiber, membrane, tubing, etc.). Support ofaffinity matrix may be made from other organic and inorganic compoundsknown to one of skill in the art, for example, polyvinylpyrrolidone(PVP), polysulfone (PS), polyethersulfone (PES), polyarylethersulfone(PAES), polyacrylate, poly(methyl methacrylate) (PMMA), poly(glycidylmethacrylate) (PGMA), poly(hydroxy metacrylate), polystyrene (PS),polytetrafluoroethylene (PTFE), polyacrylamide, polyacrolein,acrylonitrile butadiene styrene (ABS), polyacrylonitrile (PAN),polyurethane (PU), Eupergit®, polyethylene glycol (PEG),hyperfluorocarbon, agarose (i.e.cros-linked agarose), alginate,carrageenan, chitin, starch, cellulose, nitrocellulose, Sepharose®,glass, silica, kieselguhr, zirconia, alumina, iron oxide, porous carbonand mixtures and/or derivatives of said solid supports; and protonatedand deprotonated forms of this separation material.

The beads may be coated with DNA-binding proteins. DNA-binding proteinssuch as histones or anti-DNA antibodies may be immobilized by chemicallycoupling it to a solid insoluble support matrix such as polysaccharidebeads. For example, agarose beads are activated using cyanogen bromideand the cfDNA-capturing protein is incubated with the activated agaroseto allow coupling to occur. The unconjugated material is removed bywashing with buffer and the protein bound agarose is packed into thetargeted apheresis device/affinity cartridge. There are many differentmethods of chemically coupling proteins to a variety of insolublesupport matrixes. These and other matrix materials and methods ofprotein coupling known to those skilled in the art may be used in any ofthe methods and devices described herein.

For example, the attachment of a cfDNA-capturing molecule to a solidsupport can be through an amine, thiol, imide (i.e., water-solublecarbodiimide) or other chemical attachment method known to one of skillin the art to attach a polypeptide or oligonucleotide to a solidsupport.

The size of the beads can range from 30 to 200 microns, 40 to 180microns, 45 to 165 microns, 60 to 150 microns, for example. Any numberof oxidants may be used, such as sodium metaperiodate (NaIO).Alternatively, the primary hydroxyl group of cellulose can beselectively converted to yield 6-deoxy-6-carboxy-cellulose via oxidationmediated by piperidine oxoammonium salts (TEMPO) or oxidized withchlorite. See, for example, Eyle, S. and Thielemans, W., Surfacemodifucation of cellulose nanocrystals, Nanoscale, 2014, 6, 7764, DOI:10.1039/c4nr01756k) Also, cellulose (or agarose) support can be oxidizedby other compounds known to one of skill in the art, for example,chromic acid, chromium trioxide-pyridine, dimethylsulfoxide. (see, forexample, Peng, L. et al. Evaluation of activation methods with cellulosebeads for immunosorbent purification of immunoglobulins, J.Biotechnology, 5 (1987) 255-265). The oxidized beads are then incubatedwith a sufficiently purified and concentrated solution of histoneprotein, such as recombinant human histone H1.3. The reaction may bestopped and then washed with buffer to remove soluble proteincontaminants. Alternatively, the primary hydroxyl group of cellulose canbe selectively converted to yield 6-deoxy-6-carboxy-cellulose viaoxidation mediated by piperidine oxoammonium salts (TEMPO) or oxidizedwith chlorite. See, for example, Eyle, S. and Thielemans, W., Surfacemodifucation of cellulose nanocrystals, Nanoscale, 2014, 6, 7764, DOI:10.1039/c4nr01756k. Also, cellulose (or agarose) support can be oxidizedby other compounds known to one of skill in the art, for example,chromic acid, chromium trioxide-pyridine, dimethylsulfoxide. See, forexample, Peng, L. et al. Evaluation of activation methods with cellulosebeads for immunosorbent purification of immunoglobulins, J.Biotechnology, 5 (1987) 255-265).

The apheresis device may comprise a histone affinity matrix. The histoneaffinity matrix may comprise recombinant human histone H1.3. The histoneaffinity matrix may be part of an affinity column. The beads used in ahistone affinity matrix column may be cellulose beads that are oxidizedwith an oxidant. The beads can be sepharose beads, for example. Thebeads may be coated with streptavidin. The size of the beads can rangefrom 30 to 200 microns, 40 to 180 microns, 45 to 165 microns, 60 to 150microns, for example. Any number of oxidants may be used, such as sodiummetaperiodate (NaIO). Alternatively, the primary hydroxyl group ofcellulose can be selectively converted to yield6-deoxy-6-carboxy-cellulose via oxidation mediated by piperidineoxoammonium salts (TEMPO). See, for example, Eyle, S. and Thielemans,W., Surface modifucation of cellulose nanocrystals, Nanoscale, 2014, 6,7764, DOI: 10.1039/c4nr01756k) Also, cellulose (or agarose) support canbe oxidized by other compounds known to one of skill in the art, forexample: chromic acid, chromium trioxide-pyridine, dimethylsulfoxide.(See, e.g., Peng, L. et al. Evaluation of activation methods withcellulose beads for immunosorbent purification of immunoglobulins, J.Biotechnology, 1987, 5:255-265). The oxidized beads are then incubatedwith a sufficiently purified and concentrated solution of histoneprotein, such as recombinant human histone H1.3. The reaction may bestopped and then washed with buffer to remove soluble proteincontaminants.

The histone affinity matrix is prepared by a process comprising

a) oxidizing cellulose beads having a size between 100 and 250micrometers to yield activated cellulose beads;b) washing the activated cellulose beads;c) preparing a concentrated solution of recombinant human histone H1.3;d) incubating the activated cellulose beads with the concentratedsolution of recombinant human histone H1.3; ande) blocking any free CHO groups on the activated cellulose beads.

The above process may further comprise: f) washing the activatedcellulose beads with buffer.

Any oxidant may be used in step a). One exemplary oxidant is NaIO. Anymanner of washing can be undertaken in step b). For example, theactivated cellulose beads are washed with sodium bicarbonate,hydrochloric acid and water. Dialysis or other methods may be used instep c). For example, a solution of recombinant human histone H1.3 isdialyzed and the dialyzed solution is concentrated in 0.1 M NaHCO₃ at pH7-9, or at pH 8. In step d), the incubation may be performed for 3-5hours at 15-30° C., or for 4 hours at room temperature. In step e) theblocking step comprises adding 1 M ethanolamine to the activatedcellulose beads and reacting for 30 minutes to 2 hours at 15-30° C. Instep f) the activated cellulose beads, may be washed with TBS buffer.

The beads may be loaded onto a column, such as, e.g., apolytetraflouroethylene (PTFE) column. Other exemplary columns may havea wall made of polycarbonate, polyethylene, polyvinylchloride,polypropylene, polyethersulfone, polyester, or other polymer materialapproved by FDA or EMEA for manufacturing of devices for extracorporealtreating of blood or blood component.

The column, or cartridge device, can be also made of material that isnontoxic and which provides rigid support to the affinity matrix within.Typically, the material will be a plastic composition such aspolycarbonate, polyethylene, polyvinylchloride, polypropylene,polyethersulfone, polyester, polystyrene, or other similar materialapproved by the regulators such as FDA or EMEA for manufacturing ofdevices for extracorporeal treating of blood or blood component. In someembodiments, there is an inside filter at the bottom of the column(cartridge) to prevent the affinity matrix from leaving the device. Insome embodiments, there is also an inside filter at the top of thedevice to contain the affinity matrix within the device. In someembodiments, these filters are composed of plastic and/or cellulosicmaterial and have pores that will allow through passage of fluid such asplasma, but not particulate material such as affinity matrix.

In preparing a histone affinity matrix column, the histone affinitymatrix may be loaded to at least 50%, 60%, 70%, 75%, 80%, 85%, or 90%column volume. PBS, particularly cold PBS may be used to equilibrate thecolumn. Other suitable buffers may also be used to equilibrate thecolumn.

The apheresis device may comprise a lectin affinity matrix. Non-limitingexamples of useful lectins include, e.g., Galanthus nivalis (snowdrop)Lectin (GNA), Narcissus Pseudonarcissus (Daffodil) Lectin (NPA),Conconavalin A, phytohemagluttanin, and cyanovirin. In one embodiment, alectin can be coupled to an agarose affinity matrix by incubatingovernight at a neutral to slightly alkaline pH. After such incubation,extensive washing with buffer at a pH of near 7.0 to 7.5 may beundertaken to remove the unbound lectin.

A lectin affinity matrix may be prepared according to a processcomprising

a) reacting lectin with activated agarose beads to yield lectin-coupledagarose; andb) washing the lectin-coupled agarose with buffer.

The apheresis device may comprise a polyamidoamine (PAMAM) dendrimeraffinity matrix (PDAM) or polypropyleneimine (PPI) dendrimer affinitymatrix. See, e.g., Kaur et al., J Nanopart Res., 2016, 18:146.Dendrimers are unique synthetic polymers of nanometer dimensions with ahighly branched structure and globular shape. Among dendrimers,polyamidoamine (PAMAM) have received most attention as potentialtransfection agents for gene delivery, because these macromolecules bindDNA at physiological pH. PAMAM dendrimers consist of an alkyl-diaminecore and tertiary amine branches. They are available in ten generations(G0-10) with 5 different core types and 10 functional surface groups.DNA and polyamidamine (PAMAM) dendrimers form complexes on the basis ofthe electrostatic interactions between negatively charged phosphategroups of the nucleic acid and protonated (positively charged) aminogroups of the polymers. Formation of high molecular weight andhigh-density complexes depend mainly on the DNA concentration, withenhancement by increasing the dendrimer-DNA charge ratio. (Shcharbin, D.et al., Practical Guide to Studying Dendrimers. Book, iSmithers RapraPublishing: Shawbury, Shrewsbury, Shropshire, U K, 2010. 120 p. ISBN:978-1-84735-444-0.)

The PAMAM dendrimer affinity matrix prepared by a process comprising

a) washing cellulose beads with ethanol and water;b) incubating the washed cellulose beads with (±)-epichlorohydrin andNaOH to yield activated cellulose beads;c) reacting the activated cellulose beads with PAMAM dendrimer to yieldPAMAM beads and removing PAMAM dendrimer that did not react with theactivated cellulose beads; andd) blocking unconverted expoxy groups on the PAMAM beads.

The beads may be loaded onto a column, such as a polytetraflouroethylene(PTFE) column. Other exemplary columns may have a wall made ofpolycarbonate, polyethylene, polyvinylchloride, polypropylene,polyethersulfone, polyester, or other polymer material approved by FDAor EMEA for manufacturing of devices for extracorporeal treating ofblood or blood component.

An apheresis device comprising a PAMAM dendrimer affinity matrix may bemore effective at removing cfDNA, or alternatively may more completelyremove cfDNA, or alternatively may remove a greater overall amount ofcfDNA in a particular blood sample, than using an apheresis devicecomprising a histone affinity matrix and a lectin affinity matrix.

In certain embodiments, the apheresis device may comprise all of a PAMAMdendrimer affinity matrix, a histone affinity matrix and a lectinaffinity matrix.

The apheresis device may comprise an anti-DNA antibody affinity matrix.Antibodies to DNA constitute a subgroup of antinuclear antibodies thatbind single-stranded DNA, double-stranded DNA, or both (anti-ds+ss DNAantibody). They may be, e.g., IgM antibodies or any of the subclasses ofIgG antibodies. Antibodies that bind exclusively to single-stranded DNAcan bind its component bases, nucleosides, nucleotides,oligonucleotides, and ribose-phosphate backbone, all of which areexposed in single strands of DNA. Antibodies that bind double-strandedDNA can bind to the ribose-phosphate backbone, base pairs(deoxyguanosine-deoxycytidine and deoxyadenosine-deoxythymidine), orparticular conformations of the double helix (Bevra Hannahs Hahn,Antibodies to DNA. N Engl J Med 1998; 338:1359-1368). Antibodies to DNAmight also bind DNA containing supramolecular structures likenucleosomes and chromatin.

The anti-DNA antibody affinity matrix can be prepared by activatingagarose beads, such as with N-hydroxysuccinimide (NHS). The activatedbeads can then be incubated with an antibody or other reagent that hasaffinity to DNA. The excess antibodies/reagents are then removed bywashing.

An anti-nucleosome antibody affinity matrix (ANAM) prepared by a processcomprising

a) preparing activated agarose beads by crosslinkingN-hydroxysuccinimide with agarose beads;b) washing the activated agarose beads with coupling buffer comprisingNaHCO₃ and NaCl;c) adding to the coupling buffer an antibody that binds to nucleosomes,wherein the antibody is prepared in a MRL/Mp (−)+/+mouse;d) incubating the coupling buffer comprising the antibody with theactivated agarose beads to yield the anti-nucleosome antibody affinitymatrix; ande) washing the anti-nucleosome antibody affinity matrix with couplingbuffer and acetate buffer.

The apheresis device may comprise a DNA intercalator affinity matrix.There are several ways molecules can interact with DNA. Ligands mayinteract with DNA by covalently binding, electrostatically binding, orintercalating. Intercalation occurs when ligands of an appropriate sizeand chemical nature fit themselves in between base pairs of DNA.DNA-binding agents tend to interact noncovalently with the host DNAmolecule through two general modes: (i) Threading Intercalation in agroove-bound fashion stabilized by a mixture of hydrophobic,electrostatic, and hydrogen-bonding interactions and (ii) Classicalintercalation through an intercalative association in which a planar,heteroaromatic moiety slides between the DNA base pairs. Intercalativebinding, the most commonly studied, is the noncovalent stackinginteraction resulting from the insertion of a planar heterocyclicaromatic ring between the base pairs of the DNA double helix. Seehttp://nptel.ac.in/courses/104103018/35. Hoechst 33342 is abis-benzimide derivative that binds to AT-rich sequences in the minorgrove of double-stranded DNA. The heterocyclic moiety in this dye isimportant for efficiently interacting with the DNA double helix, thusmaking the Hoechst-DNA complex more stable.

The DNA intercalator affinity matrix may be prepared by oxidizing(activating) beads, such as cellulose beads (support) reacting with acompound (linker), such as N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) that link the DNA-intercalator (DNA-binding moiety,i.e. Hoechst 33342) with support surface. The beads are then washed.

A Hoechst 3342 affinity matrix prepared by a process comprising

a) oxidizing cellulose beads;b) washing the oxidized cellulose beads;c) reacting the washed oxidized cellulose beads with a solutioncomprising Hoechst 33342 and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) to yield Hoechst 33342 immobilized cellulose beads;andd) washing the Hoechst 33342 immobilized cellulose beads.

The apheresis device may comprise a hyperbranched poly-L-lysine affinitymatrix. A hyperbranched poly-L-lysine affinity matrix may be prepared bya process comprising

a) dissolving L-lysine monohydrochloride in water and neutralizing withKOH to yield an L-lysine solution;b) heating the L-lysine solution to yield a solution comprisingpoly-L-lysine;c) removing the L-lysine and salt from the solution comprisingpoly-L-lysine;d) fractionating the solution comprising poly-L-lysine to obtain afraction comprising poly-L-lysine with an average molecular weight of21,000 to 32,000;e) dialyzing and lyophilizing the fraction comprising poly-L-lysine withan average molecular weight of 21,000 to 32,000 to yield a lyophilizate;f) dissolving the lyophilizate in distilled water and dialyzing againstNaHCO₃ to yield a solution comprising HBPL; andg) incubating the solution comprising HBPL with cyanogenbromide-activated Sepharose 4B suspended in NaHCO₃.

In certain embodiments, the apheresis device may comprise all of, or anynumber of the following: a DNA intercalator affinity matrix, a Hoechst33342 affinity matrix, an anti-DNA affinity matrix, a PAMAM affinitymatrix, a histone affinity matrix, a lectin affinity matrix, and apoly-L-lysine affinity matrix.

Various apheresis procedures and methods of treatment are describedthroughout the application. Various methods and procedures comprise (a)performing an apheresis procedure comprising diverting blood or plasmafrom the patient into an apheresis device to produce purified blood orplasma; and (b) returning the purified blood or plasma with reducedlevels of the cfDNA to the patient. Any vein may be selected for optimaldiversion of the blood. For example, the blood may be diverted from theportal vein of the patient. Alternatively, the blood may be divertedfrom the femoral vein or the jugular vein of the patient.

In various embodiments of treatment, an apheresis procedure may becarried out more than once, or even twice, for example on day 1 and onday 3. If treating kidney injury, the level of kidney injury can beassessed by measuring serum creatinine and blood urea nitrogen (BUN)levels with Roche Reflotron Plus (Roche Diagnostics) before eachapheresis procedure.

Circulating cfDNA can be extracted from plasma samples with conventionalTHP (Triton-Heat-Phenol) method (Breitbach et al., PLoS ONE, 2014,9(3):e87838). Extracted cfDNA may be quantified with various assays,such as, e.g., the PicoGreen assay (Molecular Probes, Netherlands)following the manufacturer's instructions. For visualization of cfDNA inagarose gel as described in the examples, below, well known DNA dyes canbe used, including, e.g., ethidium bromide (Sigma-Aldrich), Diamond™Nucleic Acid Dye (Promega), SYBR® Gold Nucleic Acid Gel Stain (MolecularProbes). The dyes can be used as either a gel stain, a precasting agentor can be preloaded directly into sample loading buffer.

In various embodiments, performing an apheresis procedure furthercomprises separating the blood into plasma. The plasma portion may thenbe diverted to one or more affinity matrices so as to remove cfDNA.

EXAMPLES

The present invention is also described and demonstrated by way of thefollowing examples. However, the use of these and other examplesanywhere in the specification is illustrative only and in no way limitsthe scope and meaning of the invention or of any exemplified term.Likewise, the invention is not limited to any particular preferredembodiments described here. Indeed, many modifications and variations ofthe invention may be apparent to those skilled in the art upon readingthis specification, and such variations can be made without departingfrom the invention in spirit or in scope. The invention is therefore tobe limited only by the terms of the appended claims along with the fullscope of equivalents to which those claims are entitled.

Example 1: Preparation of Histone H1 Affinity Matrix and Affinity Column

The histone H1 affinity matrix and affinity column were prepared asfollows: cellulose beads (bead size of 100-250 micrometers,Sigma-Aldrich) were oxidized with sodium metaperiodate. To accomplishthis, an aqueous suspension of the beads (3 g, 5 mL) and NaIO, (0.1 g,0.5 mmol) in 10 mL of water was shaken at room temperature for 4 h. Theactivated beads were collected and washed with 1 M sodium bicarbonate,0.1 M hydrochloric acid and 200 mL of water. A solution of recombinanthuman histone H1.3 (≥98% purity, Institute of Bioorganic Chemistry,Moscow) was dialyzed and concentrated (10 mL; 5 mg/mL) in 0.1 M NaHCO₃(pH 8). Then the solution was incubated with oxidized beads (5 ml) atroom temperature for 4 h with stirring. After the incubation, 1 Methanolamine (1.5 mL) was added to the activated beads suspension (15ml) to block the free CHO groups; the reaction continued for 1 h at roomtemperature. The resulting cellulose beads with immobilized histone H1were washed three times with TBS buffer to remove soluble proteincontaminants and to provide histone H1 affinity matrix. Polycarbonatecolumns of 4 mL-30 mL volume were loaded (to 70-90% of the volume) withthe cellulose matrix with immobilized histone H1.

Example 2: Purification of the Blood of Cancer Patient from DifferentTypes of Circulating cfDNA

Separation of particle bound type of cfDNA (i.e. nucleosome-bound cfDNAand exosome-bound cfDNA) from unbound circulating cfDNA was performed asfollows: plasma from a cancer patient with advanced gastricadenocarcinoma and multiple metastases in lungs and liver (T4N2M1) wasprepared by collecting blood into citrate-treated tubes and centrifugingfor 10 minutes at 2,000 g using a refrigerated centrifuge and collectionof supernatant.

The nucleosome-bound cfDNA and exosome-bound cfDNA were removed usingtwo sequential affinity columns containing anti-histone antibody basedaffinity matrix and lectin based affinity matrix as describedrespectively in WO2007/049286A1 and U.S. Pat. No. 9,364,601.

Briefly, an anti-histone antibody affinity matrix and a column wereprepared as follows: 0.5 mL (1 volume) of streptavidin coated sepharosebeads (average bead size: 45 to 165 microns, Pierce Biotechnology, USA)were packed on to a 1.3 volume (1.3 mL) polystyrene column above glasswool. The column was equilibrated with 2 mL (4 volumes) of PBS. 1 mL(volume) of 100 μg/mL solution of biotinylated anti-histone antibodies(H2A.X; Santa Cruz Biotechnologies) were added to the column and allowedto enter the gel bed. The bottom and top caps were sequentially replacedand incubated for 2 hours at room temperature. Following incubation, thecolumn was washed with 2 mL (4 volumes) of cold phosphate bufferedsaline (PBS).

Lectin affinity matrix was prepared as follows: 2 mL (1 volume) ofLectin from Galanthus nivalis (snowdrop), i.e., GNA (Sigma-Aldrich)solution at a concentration of 10 mg/mL in 0.1M NaHCO₃, pH 9.5 was addedto 2 mL (1 volume) of CNBr activated agarose beads (Cyanogenbromide-activated-Sepharose 6 MB, 6% agarose, 200-300 μm diametermacrobeads, Sigma-Aldrich) and allowed to react overnight in the cold atpH 7.4-8.0. When the reaction was complete, the lectin coupled agarosewas washed extensively with sterile cold phosphate buffered saline (PBS)at pH 7.2-7.4. The prepared lectin affinity matrix was transferred to a0.6×6 cm polystyrene column.

For the purification from nucleosome bound cfDNA 1.0 mL of plasma wasapplied to the first affinity column (comprising anti-histone H2Aantibody affinity matrix) and allowed to flow through. Then the plasmawas applied to the second affinity (exosome binding) column (comprisinglectin [GNA] affinity matrix) and allowed to flow through.

Alternatively, the same amount of the patient plasma was allowed to flowthrough a single histone H1 affinity column prepared as described inExample 1 (cellulose beads coupled with immobilized histone H1.3).

All plasma samples were analyzed by gel electrophoresis with fluorescentDNA dye staining prior to apheresis and following the completion ofapheresis.

The electrophoretic profile of circulating cfDNA from plasma of thecancer patient prior removal of nucleosome bound DNA and exosomes (LaneA), following sequential affinity purification with anti-histone H2Aantibody and lectin affinity columns (Lane B) and following affinitypurification with histone H1.3 affinity column (Lane C) is presented inFIG. 1.

Even though nucleosome bound circulating cfDNA and exosomes were removedfrom plasma, the sample shown in the middle lane still containedsignificant amounts of circulating cfDNA visualized within a molecularrange of 100-1000 base pairs. As shown in the right lane, no DNA wasvisualized in the sample following passage through histone H1.3 affinitycolumn. Thus apheresis/purification of patient plasma through affinitymatrix containing DNA binding protein (histone 1-11.3) can remove alarge proportion of, nearly all of, or all of, nucleosome-bound cfDNA,exosome-bound cfDNA and unbound cfDNA including dsDNA, ssDNA andoligonucleotides from patient blood.

Example 3: Circulating cfDNA Purified from Nucleosome Bound DNA andExosomes Promotes Tumor Growth

60 mL of plasma was collected from a metastatic non-small-cell lungcarcinoma patient (NSCLC T3N2M+) over a few consecutive days andpurified from circulating nucleosome bound cfDNA and from exosomes usinganti-histone H2. A antibody and lectin affinity columns, consequently,as described in Example 2 (affinity matrix with anti-histone antibodiesand affinity matrix with lectin from Galanthus nivalis (snowdrop)). Foraffinity column preparation, polycarbonate 2.0×7.0 cm columns were used.Each was loaded to 70-80% of the column volume with the correspondingmatrix. The remaining circulating cfDNA was extracted from purifiedplasma using classic phenol chloroform extraction and ethanolprecipitation (Stirling, D. et al, DNA extraction from plasma and serum,In: Methods in Molecular Biology, vol. 226: PCR Protocols, SecondEdition, Ed. by J. M. C. Bartlett and D. Stirling, Humana Press Inc.,Totowa, N.J., 2003, 556 pages). Dry extracted cfDNA was stored at −70°C. The total amount of residual DNA recovered from patient plasmafollowing purification from nucleosome and exosome bound circulatingcfDNA was 9.7 μg. The cfDNA was redissolved in PBS and used for animalexperiments as described below.

The effect on tumor growth of cfDNA which was not bound to nucleosomeand exosome was tested using Panc02/C57/BL6 orthotopic model (Jiang Y-J,Lee C-L, Wang Q, et al. Establishment of an orthotopic pancreatic cancermouse model. World Journal of Gastroenterology: WJG. 2014;20(28):9476-9485). 1×10⁶ Panc02 cells suspended in ice-cold Martigelwere injected to pancreas tail of each animal (Day 0). 24 tumor bearingmice were divided into 3 groups of 8 mice each. Control group mice weregiven single daily injections of PBS (100 μL; retro-orbital venoussinus) for 10 days: from Day 10 to Day 20. Group 1 mice were given dailyinjections of 100 ng cancer patient cfDNA purified as described aboveand mice of group 2 were given with 100 ng UltraPure™ Salmon Sperm DNA(Life Technologies) with an average size of ≤2,000 base pairs (asnon-specific control) using same schedule and technique.

Table 1 below summarizes the effects of DNA injections on tumor weightin treated animals versus the control group. Tumor weight was measuredat the study termination on Day 23.

TABLE 1 Tumor Weight (g) Day 23, Group N Test Material Median ± SDControl 8 Vehicle (PBS) 1.37 ± 0.64 Group I 8 cfDNA from NSCLC T3N2M+2.53 ± 0.35 patient plasma purified from nucleosome and exosome boundcfDNA Group II 8 UltraPure ™ Salmon Sperm 1.11 ± 0.10 DNA

FIG. 2A shows tumors excised from control group mice. FIG. 2B showstumors excised from mice treated with cfDNA from an NSCLC T3N2M+ patientpurified from nucleosome and exosome bound circulating cfDNA. Tumorsfrom the control group were much smaller, dense, well separated fromadjacent organs and do not have necrosis and hemorrhages.

The circulating cfDNA from cancer patient plasma purified fromnucleosome and exosome bound circulating cfDNA retained significanttumorigenic properties. Thus, it may be beneficial to reduce levels allof nucleosome-bound cfDNA. exosome-bound cfDNA and unbound cfDNAincluding dsDNA, ssDNA and oligonucleotides.

Example 4: Preparation of Polyamidoamine Dendrimer Affinity Matrix andAffinity Column

PAMAM dendrimer affinity matrix (PDAM) and columns which contain PDAMwere prepared according to Wang (Wang, Y., et al., New method for thepreparation of adsorbent with high adsorption capacity, Chinese ScienceBulletin 2005, Vol. 50, No. 21, pp 2432-2435) as follows. Cellulosebeads (Macroporous Bead Cellulose MT 500, particle size 100-250 μm,Iontosorb, Czech Republic) were washed twice with 98% ethanol anddistilled water. 1 gram of the beads was incubated with a mixture of 1.0ml (±)-Epichlorohydrin (Sigma-Aldrich) and 3.0 ml of 2.5 M NaOH. Theactivating reaction was performed at 40° C. for 2.5 h in a shaker.Activated beads were washed thoroughly with distilled water. The epoxycontent of the resins was determined as about 0.31 mmol/g of dry beadsby titration of sodium thiosulfate with hydrogen chloride. 4.0 ml ofprepared wet activated cellulose beads was suspended with 9.0 ml of 20%solution of amino terminated (—NH₂) PAMAM dendrimer (ethylenediaminecore, generation 3.0, Sigma-Aldrich) solution and shaken at 24° C. for 5h. After the modification, unreacted PAMAM was removed by washing withdistilled water and the remaining unconverted epoxy groups on the beadswere blocked by reacting with ethylamine. The functionalized affinitymatrix was then washed with 0.1 M phosphate buffer and MilliQ water.2.0-20.0 mL of prepared affinity matrix were placed in pyrogen freepolytetraflouroethylene (PTFE) (0.5-3.0) cm×(1.0-10.0) cm column (toload of 70-90% of column volume). The prepared affinity column wassterilized by autoclaving at 121° C. for 30 min.

Example 5: Purification of Blood Plasma of Cancer Patient and StrokePatient from Different Types of Circulating cfDNA

1.0 ml aliquots of plasma samples from both an ischemic stroke patient(24 hours since stroke onset) and a cancer patient with advanced gastricadenocarcinoma with multiple metastases in lungs and liver (T4N2M1) weresubsequently purified through both anti-histone H2A antibody and lectinaffinity columns, as described in Example 2 (affinity matrix withanti-histone H2A antibodies and affinity matrix with lectin fromGalanthus nivalis (snowdrop)), or through a polyamidoamine dendrimeraffinity 0.6×10.0 cm column alone prepared as described in Example 4(affinity matrix of cellulose beads coupled with PAMAM dendrimer).

All plasma samples were analyzed by gel electrophoresis with fluorescentDNA dye staining prior to purification and following purificationcompletion,

The electrophoretic profile of circulating cfDNA. from plasma of thesepatients prior to removal of nucleosome bound cfDNA and exosomes,following affinity apheresis with anti-histone antibody and lectinaffinity columns, and following affinity purification withpolyamidoamine dendrimer affinity column are presented in FIG. 3.

Consequent purification of plasma of the cancer patient withanti-histone antibody- and lectin affinity columns removed the majorityof particle-bound circulating cfDNA; however, a visible amount ofnucleosome bound circulating cfDNA and circulating cfDNA ofmononucleosomal size and subnucleosomal size (˜below 147 base pairs inlength) remained in plasma. Plasma purification with a polyamidoamine(PAMAM) dendrimer affinity column lead to complete elimination ofcirculating cfDNA from plasma of the cancer patient. In a strokepatient, affinity purification with polyamidoamine dendrimer affinitycolumn (used as a single step) lead to sufficient elimination ofsubstantially all types of circulating cfDNA from the plasma such thatthey were undetectable.

Thus, the patient blood plasma can be purified from substantially alltypes of cfDNA, including nucleosome-bound cfDNA, exosome-bound cfDNAand unbound cfDNA. (including dsDNA, ssDNA and oligonucleotide) with anaffinity matrix containing a DNA binding polymer.

Example 6: cfDNA of Blood Plasma Purified from Nucleosome Bound DNA andExosomes has Procoagulant Activity

U.S. Pat. No. 9,642,822 discloses that high molecular weight circulatingnucleosome bound cfDNA in the form of neutrophil NETs has procoagulantactivity in patients with advanced cancer and acute vascular events. Theblood plasma of patient with stroke (24 hours since onset) and cancerpatient with advanced gastric adenocarcinoma with multiple metastasis inlungs and liver (T4N2M1) was sampled and purified consequently throughboth lectin- and anti-histone antibody affinity columns (prepared asdescribed in Example 2 (affinity matrix with anti-histone H2A antibodiesand affinity matrix with lectin from Galanthus nivalis [snowdrop]) orthrough polyamidoamine dendrimer affinity 1.0×5.0 cm column (prepared asdescribed in Example 4). Purified and untreated plasma samples werefurther defibrinated by spinning at 3,000 g for 20 min and filteringthrough a 0.22 μm filter. Samples were aliquoted into 1.0 mL plastictubes, shaken in a water bath at 50° C. for 25 min and centrifuged at10,000 g (10 min). The supernatants were stored at −80° C. and thentested in a thrombin generation assay as follows: a mixture of 25 μL ofdiluted (1:9) thromboplastin (Sigma), 25 μL of 0.9% NaCl, and 50 μL of1:1 dilution of defibrinated plasma (all reagents were diluted in 0.9%NaCl).

All reagents in the thrombin generation assay were diluted in 0.9% NaCl.A mixture of 25 μL of thromboplastin, 25 μL of 0.9% NaCl, and 50 μL of1:1 dilution of defibrinated plasma to be tested were added to wells ofa microtiter plate and prewarmed to 37° C. for 10 min. Then 50 μL of 1mM spectrozyme, a chromogenic substrate for thrombin, and 50 μL of 30 mMcalcium chloride were added sequentially. The plates were read out in anautomated enzyme-linked immunosorbent assay plate reader (Victor, PerkinElmer) at 1000 s and 405 nm at room temperature. All measurements weredone in triplicate. In this test OD value is proportional ofprocoagulant activity of plasma (thrombin generation).

TABLE 2 OD (405 nm) measured at 1000 sec Plasma sample Mean ± SD Cancerpatient, untreated  0.87 ± 0.12 Cancer patient, purified with lectin-and anti-  0.56 ± 0.08 histone antibody affinity matrices/columns Cancerpatient, purified with polyamidoamine  0.23 ± 0.07 dendrimer affinitymatrix/column Stroke patient, untreated 1.17 ± 0.4 Stroke patient,purified with lectin- and anti- 0.81 ± 0.4 histone antibody affinitymatrices/columns Stroke patient, purified with polyamidoamine 0.31 ± 0.3dendrimer affinity matrix/column Healthy donor 0.13 ± 0.2

The results are shown in Table 2. Thus, not only nucleosome- andexosome-bound circulating cfDNA but also unbound cfDNA has procoagulantactivity in cancer and acute vascular events. Thus reducing the levelsof all types of cfDNA, including nucleosome-bound cfDNA, exosome-boundcfDNA and unbound cfDNA (including dsDNA, ssDNA and oligonucleotides) isbeneficial.

Example 7: Preparation of Anti-DNA Antibody Affinity Matrix and Column

Anti-DNA antibody affinity matrix and affinity column were prepared asfollows: 5 mL of spherical beads from highly cross-linkedN-hydroxysuccinimide (NHS) activated 4% agarose, mean beads size of 90micrometers (NHS-activated Sepharose 4 Fast Flow, GE Healthcare LifeSciences) were used. The activated matrix was washed twice with cold(2-4° C.) coupling buffer (0.2 M NaHCO₃, 0.5 M NaCl, pH 8.3). 1000 μg ofhigh affinity mouse monoclonal IgM Anti-ds+ss DNA antibody ([49/4A1],ab35576, Abcam) were dialyzed against coupling buffer and then coupledaccording to the manufacturer's procedure to NHS activated Sepharose.Three cycles of washing with coupling buffer followed by 0.1 M acetatebuffer (pH 4.0) were used to remove the excess of unbound anti-DNAantibodies. 4 mL of washed affinity matrix was poured to 5 mL column andaffinity column was equilibrated in sterile Tris-HCl buffer (pH 7.4).

Example 8: Preparation of DNA Intercalator Affinity Matrix and Column

Hoechst 33342 affinity matrix and affinity column were prepared asfollows: cellulose beads (bead size of 100-250 micrometers,Sigma-Aldrich) were oxidized with sodium metaperiodate. For this aqueoussuspension of the beads (3 g, 5 mL) and NaIO, (0.1 g, 0.5 mmoL) in 10 mLof water were shaken at room temperature for 4 h. The activated beadswere collected and washed with 1 M sodium bicarbonate, 0.1 Mhydrochloric acid and 200 ml of water. 450 mg of activated cellulosebeads were mixed with 1000 mL of a pH buffered solution containing 0.047mg/mL of Hoechst 33342 (Sigma-Aldrich), and 0.4 mg/mL ofN-(3-dimethylaminopropyl)-N′-ethyl carbodiimide (EDC) and reacted at aconstant vortex rate for 1 h at 32° C. The beads with immobilizedHoechst 33342 were washed three times with deionized water to remove theunreacted dye. The prepared DNA-intercalator affinity matrix was placedinto a 4 mL volume plastic (polycarbonate) column. The column was storedat 4° C.

Example 9: Separation of Different Types of Circulating cfDNA from theBlood of Patient with Systemic Inflammatory Response Syndrome (SIRS) andMultiple Organ Dysfunction Syndrome (MODS)

Plasma was sampled from the patient admitted to the intensive care unit(ICU) diagnosed with systemic inflammatory response syndrome (SIRS) withmultiorgan failure (multiple organ dysfunction syndrome, MODS) secondaryto acute pancreatitis. Therapeutic plasma exchange was performed as arescue therapy. Aliquots of 1 mL of discharged patient plasma waspurified through both lectin and anti-histone antibody affinity columnsas described in Example 2 (affinity matrix with anti-histone antibodiesand affinity matrix with lectin from Galanthus nivalis (snowdrop)) orthrough a DNA-intercalator affinity column as described in Example 8(cellulose beads coupled with Hoechst 33342, a DNA intercalator affinitymatrix). All plasma samples were analyzed by gel electrophoresis withfluorescent DNA dye staining prior to the purification and following thepurification

As shown in FIG. 4, plasma of the SIRS patient contained significantamounts of circulating cfDNA, which gave a strong fluorescent signalfollowing staining with fluorescent DNA dye. Affinity purification withanti-histone antibody and lectin affinity columns removed nucleosomebound circulating cfDNA however a certain amount of nucleosome-boundcirculating cfDNA and circulating subnucleosomal cfDNA (˜below 147 basepairs in length) remained in plasma. Affinity purification with Hoechst33342 affinity column led to elimination of circulating subnucleosomalcfDNA but a certain amount of nucleosome-bound circulating cfDNA wasstill present. The inventors therefore tested sequential purificationwith different columns: 1 ml aliquot of the patient plasma was purifiedsequentially through Hoechst 33342 affinity column followed byanti-dsDNA antibody affinity column in a manner described in Example 2for sequential use of anti-histone antibody affinity and lectin affinitycolumns. Plasma was further checked with by gel electrophoresis withfluorescent DNA dye staining and no circulating cfDNA was detected.

Thus purification through affinity matrix containing two affinitymatrixes of present invention can remove substantially all types ofcfDNA in the patient's blood, including nucleosome-bound cfDNA,exosome-bound cfDNA and unbound cfDNA (including dsDNA, ssDNA andoligonucleotides), from blood or plasma from patient body.

Example 10: Circulating cfDNA of Plasma Purified from Nucleosome BoundDNA and Exosomes has Proinflammatory Activity and Contribute to OrganDysfunction in Sepsis

Plasma was sampled from the patient admitted to the intensive care unit(ICU) diagnosed with systemic inflammatory response syndrome withmultiple organ dysfunction syndrome (MODS) secondary to acutepancreatitis. Therapeutic plasma exchange was performed as a rescuetherapy. 100 mL of discharged patient plasma was purified through bothlectin and anti-histone antibody affinity columns (as described inExample 2, i.e. affinity matrix with anti-histone antibodies andaffinity matrix with lectin from Galanthus nivalis [snowdrop]) twice toprocure complete purification from nucleosome and exosome boundcirculating cfDNA. Remaining circulating cfDNA was extracted from theplasma purified from nucleosome and exosome as was described in Example3. The total amount of residual DNA (recovered from patient plasmapurified before from nucleosome- and exosome-bound circulating cfDNA)was about 50 μg. DNA was than resuspended in phosphate buffered saline(PBS) at pH 7.2 and used for an animal experiment as described below.

Eight 10 weeks old C57/BL6 male mice were intravenously injected with 1μg of extracted cfDNA three times with 1 h interval. Animals wereeuthanized 4 hours following the last DNA injection for collectingblood.

Plasma creatinine levels were measured by an enzymatic assay. PlasmaTNF-a, IFN-g, and IL-12 levels fluorescent magnetic bead-basedimmunoassay (Bio-Rad Laboratories, USA). Results are summarized in Table3 below.

TABLE 3 Value prior first DNA 4 h following last DNA Parameter injectioninjection. Mean ± SD Creatinine 0.063 ± 0.016 mg/dL 0.,167 ± 0.020 mg/dLIFN gamma 18.9 ± 5.4 pg/ml 46.1 ± 6.2 mg/ml TNF alpha 6.13 ± 2.5 pg/ml31.4 ± 5.4 pg/ml IL12 17.1 ± 6.2 pg/ml 278.4 ± 17.4 pg/ml

Thus, cfDNA of plasma purified from nucleosome bound DNA and exosomeshas strong proinflammatory activity and compromises organ function.

Example 11: Circulating cfDNA of Patient Plasma Purified fromNucleosome- and Exosome-Bound DNA but not Purified from Particle-FreeDNA is Responsible for TLR9 Activation

Activation of TLR9 receptors has been recently recognized as animportant component in the development of systemic host-inflammatoryresponse, organ failures, cancer invasion and metastasis, neuronalinjury in stroke, autoimmunity, eclampsia and age dependent deregulationof immunity leading to age related proinflammatory status.

The patient was a 33 year old man with acute myeloid leukemia and anHLA-matched bone marrow transplant (BMT), followed by standardimmunosuppression and antibiotic prophylaxis. About 1 month followingBMT, the patient developed erythematous rash consistent with GVHD gradeIII and severe diarrhea. Plasma samples were taken at the patient'sadmission and purified subsequently with anti-histone H2A antibody andlectin affinity columns as described in the Example 2 (affinity matrixwith anti-histone antibodies and affinity matrix with lectin fromGalanthus nivalis (snowdrop)) or purified with histone H1.3 affinitycolumn prepared as described in Example 1 (affinity matrix of cellulosebeads coupled with histone H1.3)

HEK-Blue™ hTLR9 reporter cells (Invivogen) were rinsed with medium todetach them from the culture flask and cells were resuspended to thecell density specified by the manufacturer's protocol. 180 μl of cellsuspension per well was stimulated for 24 h (37° C., 5% CO) with 60 μlof untreated patient plasma. Patient plasma was purified through bothlectin and anti-histone antibody affinity columns or purified through anH1.3 affinity column (as a single step). After incubation, analysis ofsecreted embryonic alkaline phosphatase (SEAP) was performed usingQuanti-Blue detection medium as described in the manufacturer'sinstructions. Detection of absorbance at 650 nm was measured using amicroplate reader.

TABLE 4 OD (650 nm). Plasma sample Mean ± SD Untreated sample 0.82 +0.11 Sample, puified with lectin and anti-histone 0.73 + 0.07 antibodyaffinity matrices/columns Sample purified with, histone H1.3 affinity0.21 + 0.05 matrix/column

Quantification of TLR9 activation was performed by reading the opticaldensity (OD) at 620 nm. (N=3.) The results are shown in Table 4.Surprisingly, the elimination of exosomes- and nucleosome-boundcirculating cfDNA prevented TLR9 activation by patient plasma to quitelimited extent, while removal of substantially all types ofparticle-bound and unbound cfDNA, including dsDNA, ssDNA andoligonucleotides, prevented TLR9 activation by patient plasma almostcompletely.

Example 12: Preparation of Hyper-Branched Poly-L-Lysine Affinity Matrix(PLLAM) and Affinity Column

Cationic poly-aminoacids like poly-L-lysine (PLL) are known to beefficient in condensing plasmid DNA into compact nanostructures and havebeen used for in vitro and in vivo binding of DNA.

Cationic DNA-binding polymer, namely hyper-branched poly-L-lysine (HBPL)was prepared as described in Kadlecova, Z. et al, A comparative study onthe in vitro cytotoxicity of linear, dendritic and hyperbranchedpolylysine analogs, Biomacromolecules, v. 13 (2012) pp 3127-3137): 27.45g of L-lysine monohydrochloride (reagent grade, ≥98%, Sigma-Aldrich,USA) was dissolved in 55 mL Milli-Q water and neutralized by (8.4 g KOH.Then, the solution was heated to 150° C. for 48 h under a stream ofnitrogen. Then, to remove excess salt and remaining L-lysine, thepolymerization product was dialyzed with dialysis membrane tubingagainst Milli-Q water (Snakeskin Dialysis Tubing, Thermo FisherScientific, Switzerland, molecular weight cut off: 3000 g/mol) Theproduct of dialysis was freeze-dried and then fractionated with SephadexG75 gel filtration column (GE Healthcare Life Science, Switzerland): thecolumn was loaded with 50 mL of a 2 mg/mL HBPL solution in 0.01 M HCland subsequently eluted with 0.01 M HCl. Fractions of 20 mL werecollected and lyophilized. The fraction with 21000-32000 ofweight-average molecular weight (as determined by by size exclusionchromatography) was collected and lyophilized. Lyophilized fraction wasdissolved in bidistilled water, dialysed against 0.1 M NaHCO₃ and usedfor further affinity matrix preparation. Agarose matrix which compriseimmobilized HBPL was prepared by a conventional method as follows:cyanogen bromide-activated Sepharose 4B (wet weight 10 g, Sigma) wassuspended in 10 ml of 0.1M NaHCO₃, mixed with 10 ml of 21000-32000 HBPLfraction (5 mg/ml in 0.1 M NaHCO₃), and stirred for 24 h at 4° C. Theprepared HBPL Sepharose (4 mg of HBPL per ml bead suspension) was thenpoured in a polycarbonate column (1.0×12 cm) and washed with 750 ml of0.1 M NaHCO₃, 750 ml of 0.5 M NaCl and adjusted to pH 9.2. The columnwas equilibrated with 0.05 M Tris-HCl buffer, pH 7.5. The preparedaffinity column with hyper-branched poly-L-lysine affinity matrix(PLLAM) was stored at 4° C.

Example 13: Separation of Different Subtypes of Circulating cfDNA fromthe Blood of Patient with Neurodegenerative Disease

Circulating cfDNA from patients with neurodegenerative disorders canpass through the blood brain barrier (BBB) and induce neuronal celldeath. The use of deoxyribonuclease enzyme could abolish this effect.See Int. Pat. Appl. Pub. WO2016190780. To investigate the effect ofdifferent subtypes of circulating cfDNA on neuronal cell death and tosee if purification of blood from all of nucleosome bound cfDNA, exosomebound cfDNA and unbound cfDNA including dsDNA, ssDNA andoligonucleotides might prevent neuronal cell death, the followingexperiments were performed.

For neuronal cultures, cerebral cortices were removed from embryonic day(E) 15-17 Sprague Dawley rat embryos. Cortical explants were dissectedinto pieces of about 200-400 μm² using fine needles and dissociated withthe Papain Dissociation System (Worthington Biochemicals) according tothe manufacturer's instructions and further kept on ice-cold minimumessential medium (Gibco). Neurons were plated on 13 mm diameter glasscoverslips coated first with poly-D-lysine (10 μg/mL in PBS) followed bylaminin (10 μg/mL in PBS) (Gibco) and cultured for 24 hrs. at 37° C. ina humidified 8% CO2 (v/v) atmosphere for 24-48 hrs. in neurobasal mediumwith 1% (v/v) Antibiotic-Antimycotic (Gibco).

After an initial period of culturing the cell culture media was dilutedtwice (v/v) with one of the following plasma samples with furtherculturing for another 24 hrs: (a) plasma of a healthy 20 year old donor,(b) plasma of the patient with rapidly progressed Alzheimer's disease(AD), (c) plasma of the same AD patient treated for 6 hours with 5 μg/mLof DNase I (Pulmozyme, Genentech), (d) plasma of the same AD patientfollowing passage through both of lectin and anti-histone H2A antibodyaffinity columns (prepared as described in Example 2, i.e. affinitymatrix with anti-histone H2A antibodies and affinity matrix with lectinfrom Galanthus nivalis [snowdrop]), and (e) plasma of the same ADpatient following passage through histone H1.3 affinity column (thematrix was prepared as described in Example 1, i.e. affinity matrix ofcellulose beads coupled with histone H1.3) and placed to 0.8×9 cmpolycarbonate column (up to 80% of column volume), with the volume ofplasma samples passed through the corresponding columns being about 2.0mL.

The electrophoretic profile of circulating cfDNA. from plasma samplesused in cell culture experiments are presented in FIG. 5.

Only a limited amount of nucleosome-bound circulating cfDNA in the formof mononucleosomes was detected in the plasma of a healthy donor. Highlevels of nucleosome bound circulating cfDNA in the form of mono andoligonucleosomes were detected in the plasma of an AD patient. Treatmentof AD patient plasma with DNase I enzyme resulted in a decrease of DNAcontent in oligonucleosomal and mononucleosomal fractions, but with asignificant increase of DNA in subnucleosomal fraction (˜below 147 basepairs in length). Plasma of an AD patient purified with lectin andanti-histone H2A antibody affinity columns did not containnucleosome-bound circulating cfDNA but only subnucleosomal (i.e.,unbound) cfDNA. Plasma of an AD patient treated with histone H1.3affinity column (as a single step) did not contain circulating cfDNA.

Induction of apoptic cell death marker Caspase 3 was determined indissociated cortical neurons cultured following 24 hours of exposure toplasma samples. Cells were fixed in 4% (w/v) paraformaldehyde (PFA) andincubated for 1 hour with cleaved Caspase 3 antibody (Abcam) diluted1:500 in PBS. Cells were washed and incubated for 1 hour with goatanti-rabbit polyclonal Alexa Fluor 488 antibodies (Invitrogen) in PBSprior to washing and counting.

TABLE 5 % of cells positive for Caspase 3; median for three repetitivePlasma sample cell cultures Healthy 20 Y donor sample, untreated 5.3% ADpatient sample, untreated 30.0% AD patient sample treated with DNase I15.7% AD patient sample purified with lectin and anti- 17.7% histoneantibody affinity matrices/columns AD patient sample purified with H1affinity 7.7% matrix/column

The results are shown in Table 5. Thus, purification of blood fromsubstantially all types of cfDNA, including nucleosome-bound cfDNA,exosome-bound cfDNA and unbound cfDNA (including dsDNA, ssDNA andoligonucleotides) prevents neuronal cell death to substantially higherextent than a simple purification from nucleosome-bound cfDNA andexosome-bound cfDNA and even better than cleavage of circulating cfDNAin plasma with DNase I enzyme, probably due to release of byproducts ofDNA enzymatic degradation or low sensitivity of circulating cfDNA toDNase I.

Example 14: Reactivation of Endogenous Deoxyribonuclease

Deoxyribonuclease enzyme (DNase) is the principal enzyme responsible fordegradation of high molecular weight DNA in circulation. Multiplestudies show that deoxyribonuclease activity is suppressed in certainconditions involving raise of circulating cfDNA in blood, such ascancer, metastatic cancer, autoimmune disease, sepsis, infertility,(Tamkovich S N, Circulating DNA and DNase activity in human blood. AnnNY Acad Sci. 2006 September; 1075:191-6; Martinez-Valle, DNase 1activity in patients with systemic lupus erythematosus: relationshipwith epidemiological, clinical, immunological and therapeuticalfeatures. Lupus. 2009 April; 18(5): 418-23; EP20070827224; Travis JGould, Cellular and Biochemical Properties of Cell-Free DNA: APrognostic Marker In Severe Sepsis Patients, Blood 2011, 118:2169)

To assess how reduction of nucleosome-bound cfDNA, exosome-bound cfDNAand unbound cfDNA including dsDNA, ssDNA and oligonucleotides affectsDNaseI activity plasma the following experiment was performed. cfDNA wasmeasured in plasma using method described by Goldstein (Goldshtein, H.et al., A rapid direct fluorescent assay for cell-free DNAquantification in biological fluids, Annals of Clinical Biochemistry,Vol 46, Issue 6, pp. 488-494). SYBR® Gold Nucleic Acid Gel Stain,(Invitrogen) was diluted first at 1:1000 in dimethyl sulphoxide and thenat 1:8 in phosphate-buffered saline. 10 μL of plasma samples wereapplied 96-well plates. 40 μl of diluted SYBR Gold was added to eachwell (final dilution 1:10,000) and fluorescence was measured with a 96well fluorometer at an emission wavelength of 535 nm and an excitationwavelength of 485 nm.

DNase I western blotting was performed in plasma samples separated using10% SDS-PAGE gels, transferred onto polyvinylidene difluoride (PVDF)blotting membranes, and incubated with goat anti-human DNase Iantibodies (Santa Cruz Biotechnology). Binding was visualized usingSuperSignal Chemiluminescent Substrate (Pierce) after incubation withHRP-conjugated anti-goat IgG.

Serum deoxyribonuclease activity was measured using ORG590 (Orgentec)according to the manufacturer's protocol. Detection was performed usingmicroplate photometer (Multiscan FC) at 450 nm with a correctionwavelength of 620 nm.

Blood was sampled from 56-year-old female patient with breast cancer,multiple metastasis in lungs, liver and medlastenum (T4N3M1). 5 mL,plasma aliquote was subjected to multiple runs through 1 mLpolycarbonate column (0.5×5 cm) containing 0.5 mL of histone H1.3affinity matrix: assessment of electrophoretic profile of circulatingcfDNA, DNAse western blot and quantification of deoxyribonucleaseactivity and circulating extracellular content were measured after eachcolumn run. The results are summarized in FIG. 6.

The electrophoretic assessment of circulating cfDNA profile showed acontinuous decrease of all fractions content alongside with increasingnumber of column runs. That observation was confirmed by directquantification of circulating cfDNA in plasma. A comparable amount ofDNase I enzyme as detected by western blot was present in patient plasmainitially. However enzymatic activity of DNase I was heavily suppressedand became meaningful only after 4 column runs when the amount ofcirculating cfDNA was decreased approximately twice.

Thus the apheresis according current invention wherein the overallcirculating levels of cfDNA in said mammal is reduced by at least 50%might reactivate the activity of endogenous DNase I enzyme, which isbeneficial for patients who require lowering of circulating cfDNAlevels.

Based on highest reported levels of circulating cfDNA of approximately5000 ng/mL (which are reported for some advanced cancer, septic patientsand patient with trauma) the affinity column or combination of affinitycolumns with binding capacity of 30 mg would be able to provide almostcomplete purification of patient plasma from all of nucleosome boundcfDNA, exosome bound cfDNA and unbound cfDNA including dsDNA, ssDNA andoligonucleotides.

Example 15: Preparation of an Affinity Column that ContainsAntinucleosome Antibody Affinity Matrix (ANAM)

A mouse monoclonal nucleosome-specific antibody was prepared usingMRL/Mp (−)+/+ mouse according to the method described in M. J. Losman,Monoclonal autoantibodies to subnucleosomes from a MRL/Mp (−)+/+ mouse.Oligoclonality of the antibody response and recognition of a determinantcomposed of histones H2A, H2B, and DNA. J Immunol Mar. 1, 1992, 148 (5)1561-1569). Prepared monoclonal (IgG) antibodies (mAbs), named here asAN-1 and AN-44, correspondingly, were selected on the basis of theirability for selective binding of nucleosomes but not components ofnucleosomes like core histones or DNA. (Kees Kramers, Specificity ofmonoclonal anti-nucleosome auto-antibodies derived from lupus mice,Journal of Autoimmunity, V. 9, Issue 6, 1996, P. 723-729). The relativeaffinity of AN-1 and AN-44 to nucleosomes and histone and non histonecomponents of nucleosome are summarized in Table 6 below.

TABLE 6 MAbs AN-1 AN-44 Nucleosome 17,400 12,000 DNA 200 300 HistonesH2A/H2B <10 <10 Histones H3/H4 <10 <10

1 mL HiTrap NHS activated HP column prepacked with NHS activatedSepharose High Performance (GE Healthcare) was used for affinitymatrix/cartridge preparation. 200 μg of AN-1 were coupled according tothe manufacturer's procedure to NHS activated Sepharose

Based on the affinities data presented at the table above it is obviousthat ANAM binds only nucleosome-bound circulating cfDNA but not unboundcfDNA, including dsDNA, ssDNA and oligonucleotides.

Thus, in order to secure binding of unbound cfDNA, including dsDNA,ssDNA and oligonucleotides, in animal experiment two sequential columnswere used. One column with anti-nucleosome antibody affinity matrix(ANAM) was prepared as described above in this example. A second columnwith polyamidoamine dendrimer affinity matrix was prepared as describedin Example 4.

Example 16: Apheresis Procedure

Chronic venous catheters were inserted into the femoral vein and thevena jugulars of experimental rats under general anesthesia (i.p.injection of 0.8 mg xylazine and 4 mg ketamine). Catheters were flushedthree times per week with heparinized saline during the study. Beforeeach apheresis procedure, a heparin bolus was given (90 IU/100 g bodyweight (b.w.). The extracorporeal system was fully filled withheparinized saline and thereafter, the catheter endings were connectedwith the extracorporeal system.

For animal apheresis experiments, the affinity columns described in thisspecification were fitted with inlets and outlets for further embeddingthese prepared affinity columns to second (plasma) circuit ofextracorporeal/apheresis system.

In the first circuit of the system, blood was pumped (Rotary peristalticMini-pump, Fisher Scientific) from the animal (femoral vein) via aplasma separator (Saxonia medical, Radeberg, Germany) and returned tothe animal by a venous catheter. The separated plasma entered the secondcircuit (through second Rotary peristaltic Mini-pump) and passed throughone or more affinity cartridges according to the specific examples ofthe apheresis procedures described in this specification.

In the first circuit of the system, blood was pumped (Rotary peristalticMini-pump, Fisher Scientific) from the animal (femoral vein) via aplasma separator (Saxonia medical, Radeberg, Germany) and returned tothe animal by a venous catheter inserted to jugular vein. The separatedplasma entered the second circuit (supported by second Rotaryperistaltic Mini-pump) and passed through affinity cartridges (accordingto the specific examples of the apheresis procedures described in thisspecification), and returned to animal body via polymer line alsoconnected to the catheter inserted into jugular vein.

Example 17: Apheresis Treatment of Sepsis and Septic Kidney Injury

Classic sepsis-induced models by the method of cecal ligation andpuncture (CLP) were established. Female Sprague-Dawley (SD) rats of350-400 g body weight were used. Animals were anesthetized with sodiumpentobarbital (50 mg/kg intraperitoneally).

A midline abdominal incision about 1.5 cm was performed. The cecummesentery was dissected to expose the cecum. Then, the cecum was ligatedbetween the terminal and ileocecal valve so that intestinal continuitywas maintained. Then, the cecum was perforated by singlethrough-and-through puncture with a 21-gauge needle in the centralsegment of ligation. The tied segment was gently pressed to ensure thata small amount of feces was extruded on to the surface of the bowel. Thececum was returned to the abdominal cavity. The surgical wound wassutured layer by layer with absorbable suture for the muscle layer andwith surgical staples for the skin. After operation, the rats wereinjected with 10 ml/kg warm 0.9% sodium chloride for injection and afterrecovery randomly the animals were randomly divided into four groups(Groups 1-3; 6 animals in each group) according to the treatment.

The apheresis treatment was performed as was described in Example 16.The apheresis procedure was carried out twice: on day 1 (24 hrs. afterCLP), and day 3 (72 hrs. after CLP). 6 rats get apheresis procedureusing column/cartridge with antinucleosome antibody affinity matrix(ANAM) prepared as specified in Example 15, and 6 rats get apheresisprocedure using column/cartridge containing PAMAM dendrimer affinitymatrix (PDAM) prepared as specified in Example 4. Six rats (negativecontrol group) get apheresis procedure with cartridge that was loadedwith corresponding volume of unmodified support cartridge). Level ofacute kidney injury (renal function) was assessed by measurement ofserum creatinine and blood urea nitrogen (BUN) levels with RocheReflotron Plus (Roche Diagnostics before each apheresis procedure.Circulating cfDNA was extracted from 100 μL plasma samples withconventional THP (Triton-Heat-Phenol) method (Breitbach et al., PLoSONE, 2014, 9(3):e87838). DNA was quantified with the PicoGreen assay(Molecular Probes, Netherlands) following the manufacturer'sinstructions and cfDNA changes were expressed as percentage of DNA levelto baseline i.e. to the level before first apheresis procedure. Fornegative control group the columns/cartridges containing correspondingamount of unmodified support (Macroporous Bead Cellulose MT 500,particle size 100-250 μm, Iontosorb, Czech Republic, washed twice with98% ethanol and bidistilled water) were prepared. The survival rate (120hours after CLP) was assumed as main parameter of treatment efficacy ofsepsis. The allocation of the animals and the results are shown in Table7, below.

TABLE 7 Hours post CLP 24 48 72 96 120 CLP + apheresis with unmodifiedsupport cartridge (Negative Control); n = 6 Circulating levels of 100%/100% 137%/137% cfDNA (before/after apheresis) Serum creatinine,140 ± 20 160 ± 12 194 ± 31 215 ± 16 μmol/L Blood urea nitrogen 11.2 ±2.1 14.9 ± 2.8 16.8 ± 3.5 18.2 ± 3.0 (BUN), mmol/L Survival 1 of 6 CLP +apheresis with ANAM cartridge; n = 6 Circulating levels of 100%/58%66%/32% cfDNA (before/after apheresis) Serum creatinine, 136 ± 12 139 ±13 145 ± 14 140 ± 13 μmol/L Blood urea nitrogen 12.3 ± 2.2 12.8 ± 2.213.5 ± 2.5 14.7 ± 2.1 (BUN), mmol/L Survival 3 of 6 CLP + apheresis withPDAM cartridge, n = 6 Circulating levels of 100%/21% 33%/12% cfDNA(before/after apheresis) Serum creatinine, 138 ± 12 100 ± 13 105 ± 14111 ± 16 μmol/L Blood urea nitrogen 12.4 ± 1.7  8.4 ± 1.0 10.4 ± 2.212.6 ± 3.7 (BUN), mmol/L

The results show that the PDAM apheresis device was able to capturesubstantially all types of of cfDNA, including nucleosome-bound cfDNA,exosome-bound cfDNA and unbound cfDNA (including dsDNA, ssDNA andoligonucleotides) and provided a better therapeutic efficacy and moreefficiently reduced the level of circulating cfDNA in sepsis and septickidney injury.

Example 18: Apheresis Treatment of Chemotherapy Related Toxicity Signs

18 female Sprague-Dawley (SD) rats of 300-350 g body weight wereprepared for apheresis procedure as described in Example 16 and receivedsingle intravenous bolus injection of paclitaxel (Taxol, Bristol-MyersSquibb S.r.L.) at 10 mg/kg dose. The apheresis procedure was started 4hours following paclitaxel injection and continued for 12 hours; 6 ratsget apheresis procedure using column/cartridge with antinucleosomeantibody affinity matrix (ANAM), and 6 rats get apheresis procedureusing column/cartridge containing hyper-branched poly-L-lysine affinitymatrix (PLLAM). 6 rats (negative control) get apheresis procedure withcartridge that was loaded with corresponding volume of unmodifiedsupport (Sepharose 4B).

Circulating cfDNA levels were quantified and presented as described inExample 17 (with cfDNA expressed as percentage of DNA level tobaseline). The survival rate (24 hours after bolus) was assumed as mainparameter of treatment efficacy. The allocation of the animals and theresults are shown in Table 8 below.

TABLE 8 Hours post Paclitaxel bolus 4 h 16 h 24 h Paclitaxel + apheresiswith unmodified support cartridge (Negative Control) n = 6 Circulatinglevels of 100% 230% cfDNA (before/after apheresis) Survival zero from 6Paclitaxel + apheresis with ANAM cartridge; n = 6 Circulating levels of100% 165% cfDNA (before/after apheresis) Survival 2 from 6 Paclitaxel +apheresis with sequential PLLAM cartridge, n = 6 Circulating levels of100%  65% cfDNA (before/after apheresis) Survival 5 from 6

The results show that PLLAM apheresis device was able to capturesubstantially all types of cfDNA, including nucleosome-bound cfDNA,exosome-bound cfDNA and unbound cfDNA (including dsDNA, ssDNA andoligonucleotides) provide better protection/therapeutic efficacy andmore efficiently reduced the level of circulating cfDNA in animalspoisoned by a chemotherapeutic drug.

Example 19. Purification/Apheresis of Plasma cfDNA with One Cartridgethat Captures Nucleosome- and Exosome-Bound DNA and Another Cartridgethat Captures Unbound cfDNA Including dsDNA, ssDNA and Oligonucleotides

For the measurements of plasma cfDNA level, cfDNA was extracted from 500μL plasma samples using modified HTP method (Xue, X., et al. Optimizingthe yield and utility of circulating cell-free DNA from plasma andserum, Clinica Chimica Acta, V. 404 (2009), pp. 100-104) and quantifiedusing the PicoGreen assay (Molecular Probes, Netherlands) according tothe manufacturer's instructions.

When cfDNA was undetectable in a sample by PicoGreen assay, the absenceof cfDNA in the samples was further confirmed by DNA electrophoresis inagarose gel in a manner described in the specification above.

For apheresis/purification procedures plasma samples were graduallyapplied to the corresponding affinity columns and allowed to flowthrough.

A 2.0 mL plasma sample obtained from 67-year-old septic shock patientwas purified consequently through an ANAM affinity column. The ANAMaffinity column (which captures nucleosome-bound cfDNA) was prepared onthe basis of 1 mL HiTrap NHS activated HP column (as described inExample 15) and lectin affinity column (which captures exosome-boundcfDNA) was prepared as described in the Example 2 (affinity matrix withlectin from Galanthus nivalis [snowdrop]).

Initial cfDNA level in patient plasma was 1150 ng/mL with significantpresence of substantially all types of cfDNA visualized by DNAelectrophoresis in agarose gel (See FIG. 7, lane A). The level of cfDNAin plasma following first run through combination of ANAM and lectinaffinity columns has decreased to 350 ng/mL. Partially purified patientplasma was further subjected to second run through same combination offresh ANAM and lectin affinity columns. The level of cfDNA in plasmafollowing second run remained unchanged with visible amounts of cfDNA ofnon-nucleosomal origin with molecular weight of up to 750 kDa visualizedby DNA electrophoresis in agarose gel with fluorescent DNA dye staining(See FIG. 7, lane C).

The experiment made clear that inability of ANAM and lectin affinitycolumns to completely purify patient plasma from cfDNA did not relate tooverall binding capacity of the AMAM and lectin affinity columnscombination but rather to its inability to capture cfDNA ofnon-nucleosomal or non-exosomal origin from patient plasma. In order toconfirm this we further purified the sample through anti-DNA antibodyaffinity column prepared as described in Example 7 (matrix of agarosecoupled with high affinity mouse monoclonal IgM Anti-ds+ss DNA).Following one purification run the level of cfDNA in patient plasmabecame undetectable as measured by PicoGreen assay. This observation wasfurther confirmed by absence of visible DNA material following DNAelectrophoresis in agarose gel.

Thus, use of two sequential affinity columns/cartridges, wherein onecolumn/cartridge captures nucleosome-bound DNA and exosome-bound DNA andanother column/cartridge captures unbound cfDNA including dsDNA, ssDNAand oligonucleotides, is very effective for purification/apheresis ofpatient blood from all type of circulating cfDNA.

Another 2 mL plasma sample from the same patient were purifiedconsequently through DNA-intercalator affinity column (prepared asdescribed in Example 8, i.e., cellulose beads coupled with Hoechst33342) and anti-DNA antibody affinity column (prepared as described inExample 7, i.e., matrix of agarose coupled with high affinity mousemonoclonal IgM anti-DNA). The level of cfDNA in plasma following firstrun through combination of DNA-intercalator and anti-DNA antibodyaffinity columns has decreased to 475 ng/mL with cfDNA of differentorigin visualized by DNA electrophoresis in agarose gel (FIG. 7, LaneB). This partially purified patient plasma was further subjected to asecond run through the same combination of fresh DNA-intercalator andanti-DNA antibody affinity columns. The level of cfDNA in plasmafollowing the second run in the patient plasma became undetectable asmeasured by PicoGreen assay. This observation was further confirmed byabsence of visible DNA material following DNA electrophoresis in agarosegel with fluorescent DNA dye staining.

Thus, the use of a combination of columns/cartridges containing matriceswhich bind substantially all types of cfDNA (including nucleosome-boundcfDNA, exosome-bound cfDNA and unbound cfDNA [including dsDNA, ssDNA andoligonucleotides]) according to the invention permits capture of anunusually high amount of cfDNA.

Example 20. Purification/Apheresis of Plasma from the Portal Vein toPurify cfDNA from Blood of Rats with Acute Pancreatitis

Six male Sprague-Dawley rats, 250-350 grams were used in the experiment.All surgical procedures were performed on a heated operating table undergeneral anaesthesia with i.p. injection of 0.8 mg xylazine and 4 mgketamine.

Acute pancreatitis was induced as follows. During laparotomy the papillaof Vater was cannulated transduodenally using a 24G Abbocath®-T i.v.infusion cannula. Before pressure monitored infusion of 0.5 mLsterilized glycodeoxycholic acid in glycylglycine-NaOH-buffered solution(10 mmol/L, pH 8.0, 37° C.), the common bile duct was clamped and bileand pancreatic fluid were allowed to drain through the cannula. Directlyafter infusion, hepato-duodenal bile flow was restored by removal of theclamp. The puncture hole in the duodenum was carefully closed using an8.0 polyprolene serosal suture.

After closure of the abdomen in Rats 1, 2 and 3, chronic venouscatheters were inserted into the femoral vein and the jugular vein asdescribed in Example 16.

Rats 4, 5 and 6 had a portal vein catheter implanted into the hepaticportal vein 8 mm caudal of the liver as described by Strubbe (Strubbe J.H. et al, Hepatic-portal and cardiac infusion of CCK-8 and glucagoninduce different effects on feeding. Physiol Behav 46: 643-646, 1989).

The apheresis treatment was performed as described in Example 16 usingPDAM affinity cartridge, prepared as was described in Example 4 andfitted with polypropylene inlet and outlet. The apheresis procedure wascarried out daily during days 1-3 with 12 hours duration of eachapheresis procedure.

The survival rate (96 hours following induction of pancreatitis) wasassumed as a main parameter of treatment efficacy. For quantification ofcfDNA of rat and cfDNA of bacteria origin (i.e., bacterial load), totalcfDNA was isolated from 200 μL rat plasma samples using a QIAamp DNAMini Kit according to the manufacturer's instructions. The survival rate(96 hours following induction of pancreatitis) was assumed as a mainparameter of treatment efficacy. For quantification of cfDNA of rat andcfDNA of bacteria origin (i.e., bacterial load), total cfDNA wasisolated from 200 μL rat plasma samples using a QIAamp DNA Mini Kitaccording to the manufacturer's instructions. cfDNA concentration on theplasma samples were measured by quantitative polymerase chain reaction(PCR) using the ABI PRISM 7700 Sequence Detector (Applied Biosystems)and TaqMan Universal PCR Master Mix (Applied Biosystems) according tothe manufacturer's protocol. For quantification of cfDNA of bacterialorigin, specific primers and a probe were designed for the conservedregions of bacterial 16S rDNA: the forward primer,5′-TCCTACGGGAGGCAGCAGT-3′, the reverse primer5′-GGACTACCAGGGTATCTAATCCTGTT-3′ and the probe(6-FAM)-5′-CGTATTACCGCGGCTGCTGGCAC-3′-(TAMRA) (see: Mangala, A.;Nadkarni, A. Determination of bacterial load by real-time PCR using abroad-range (universal) probe and primers set. Microbiology, 2002, vol.148, pp. 257-266). TaqMan Gene Expression Assay rat β-actinRn00667869_ml (Applied Biosystems) was used for amplification of ratgenomic cfDNA

Survival/outcome and the results of each PCR (Ct, i.e. threshold cyclevalue) for rat β actin gene and bacterial 16S rDNA in blood plasmasampled from jugular vein are presented in Table 9.

TABLE 9 Vein from which the blood was β actin gene Survival/ diverted.Ct* 16S rDNA Ct* Outcome Rat 1 Femoral vein 29.49 ± 0.161 24.22 ± 0.096Alive at 96 h Rat 2 Femoral vein  29.2 ± 0.379 23.85 ± 0.218 Dead at 82h Rat 3 Femoral vein 28.62 ± 0.278 23.59 ± 0.109 Alive at 96 h Rat 4Portal vein 30.26 ± 0.176 27.89 ± 0.112 Alive at 96 h Rat 5 Portal vein30.26 ± 0.21  25.78 ± 0.155 Alive at 96 h Rat 6 Portal vein 30.44 ±0.151 29.42 ± 0.341 Alive at 96 h *Mean ± SD of three independent runs.Ct values are natural logarithmic and inverse to the amount of nucleicacid or gene of interest in the sample. The Ct is the cycle number atwhich the fluorescence generated within a reaction crosses the thresholdline.

The results show that diverting/removing the blood for apheresis into anapheresis device according to the invention from portal vein resulted inbetter (as compared to diverting of the blood from femoral, i.e.,non-regional vein) survival and more effective purification of bloodfrom cfDNA (including cfDNA of bacterial origin) in rats with acutepancreatitis.

Thus, in clinical circumstances where the pathological processresponsible for the release of cfDNA (tumor growth, septic or asepticinflammation, bacterial DNA release) originates from areas/regionsdrained primarily by portal vein (esophagus, gastric, intestinal,splenic, pancreatic, gallbladder, peritoneal cavity) diverting the bloodfor apheresis procedure from the portal vein might be beneficial.

Example 21. Comparison of cfDNA Removal from Patient Plasma by HistoneH1 Affinity Matrix, PAMAM Dendrimer Affinity Matrix and Poly-L-LysineAffinity Matrix (PLLAM)

Poly-L-lysine affinity matrix (PLLAM) was produced as specified inExample 12.

PAMAM dendrimer affinity matrix (PDAM) was produced as specified inExample 4. Histone H1 affinity matrix was produced as specified inExample 1.

Model plasma enriched with cfDNA was produced by mixing of plasma ofhealthy volunteer with marker DNA (lkbp plus DNA Ladder, Invitrogen) tothe final cfDNA concentration of 10 μg/ml. Adsorption capacity ofpoly-L-lysine affinity matrix (PLLAM), PAMAM dendrimer affinity matrix(PDAM) and histone H1 affinity matrix with respect to model plasmaenriched with artificial 1 kbp plus DNA Ladder was tested by volumeadsorption method with affinity matrix:plasma ratio 1:5 (100 μl ofaffinity matrix was mixed with 500 μl of model plasma) for 1 hour at 37°C. under slow rotation. Ethanolamine Sepharose FF was used as a control.Plasma samples were analyzed by 1% agarose gel electrophoresis usingE-Gel Invitrogen system prior to incubation and upon sedimentation ofaffinity matrix. cfDNA was extracted from patient plasma using QIAampDNA Blood Mini Kit, Quagen and quantified with Qubit 3.0 fluorimeter.Same affinity matrixes were incubated with plasma of the patientdiagnosed with odontogenic-related sepsis with affinity matrix:plasmaratio 1:10 (100 μl of affinity matrix was mixed with 1 ml of patientplasma) using the same incubation conditions. In one hour, affinitymatrix was removed by centrifugation. Plasma samples were analyzed by 1%agarose gel electrophoresis using E-Gel Invitrogen system prior toincubation and upon sedimentation of affinity matrix. EthanolamineSepharose FF was used as a control. cfDNA was extracted from patientplasma using QIAamp DNA Blood Mini Kit, Quagen and quantified with Qubit3.0 fluorimeter. cfDNA quantification data are presented in Table 10,below.

TABLE 10 cfDNA content in model plasma; ng/ml, median ± SD Prior toAfter incubation incubation Control H1.3 PDAM PLLAM Model plasmaenriched  992 ± 24  942 ± 17 44 ± 19  92 ± 23  83 ± 24 with cfDNA Plasmafrom patient 1832 ± 43 1648 ± 17 57 ± 17 488 ± 24 392 ± 43 withodontogenic- related sepsis

It appears that poly-L-lysine affinity matrix (PLLAM), PAMAM dendrimeraffinity matrix (PDAM) and histone H1 affinity matrix have equalcapacity to remove model cfDNA from model plasma enriched with cfDNA,but histone H1 affinity matrix is significantly superior in removingcfDNA from patient plasma. The finding was confirmed by electrophoreticanalysis of the samples (see FIGS. 8 and 9).

Volume adsorption test with of poly-L-lysine affinity matrix (PLLAM),PAMAM dendrimer affinity matrix (PDAM) and histone H1 affinity matrixyielded almost same electrophoretic picture with marginal cfDNA content.

Volume adsorption test with poly-L-lysine affinity matrix (PLLAM), PAMAMdendrimer affinity matrix (PDAM) and histone H1 affinity matrix yieldeddifferent electrophoretic picture with marginal cfDNA content followingincubation with histone H1 affinity matrix but meaningful cfDNA contentfollowing incubation with poly-L-lysine affinity matrix and PAMAMdendrimer affinity matrix.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims. It is further to be understood that allvalues are approximate, and are provided for description.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

1. A device configured to perform apheresis comprising one or moreaffinity matrices, wherein said one or more affinity matrices arecapable of capturing nucleosome-bound cell free DNA (cfDNA),exosome-bound cfDNA, and unbound cfDNA from blood or plasma of asubject.
 2. The device of claim 1, wherein the unbound cfDNA comprisesdsDNA, ssDNA and oligonucleotides.
 3. The device of claim 1, wherein thedevice comprises two or more affinity matrices.
 4. The device of claim3, wherein (i) the first one or more affinity matrices is capable ofcapturing nucleosome-bound cell free DNA (cfDNA) and/or exosome-boundcfDNA and (ii) the second one or more affinity matrices is capable ofcapturing unbound cfDNA, and wherein the first and second affinitymatrices are arranged within the device in any order.
 5. The device ofclaim 4, wherein (i) the first one or more affinity matrices comprises aDNA binding protein, an anti-histone antibody, an anti-nucleosomeantibody, a DNA intercalating agent, a DNA binding polymer, an anti-DNAantibody, a lectin, or and any combination thereof, and (ii) the secondone or more affinity matrices comprises a DNA binding protein, a DNAintercalating agent, a DNA binding polymer, an anti-DNA antibody, or andany combination thereof. 6.-25. (canceled)
 26. The device of claim 1,wherein the device comprises a single affinity matrix.
 27. (canceled)28. The device of claim 27, wherein the histone is H1 histone.
 29. Thedevice of claim 28, wherein the histone is H1.3 histone.
 30. The deviceof claim 1, wherein the one or more affinity matrices matrix comprises aDNA binding polymer, a DNA binding protein, an anti-histone antibody, ananti-nucleosome antibody, a DNA intercalating agent, an anti-DNAantibody, a lectin, or any combination thereof. 31.-39. (canceled)
 40. Amethod of reducing the level of cell free DNA (cfDNA) in the blood of asubject, the method comprising: (a) performing an apheresis procedurecomprising diverting blood or plasma from the subject into the apheresisdevice of claim 1 to produce blood or plasma with reduced levels of thecfDNA; and (b) returning the blood or plasma with reduced levels of thecfDNA to the subject, wherein the apheresis procedure reduces the levelof nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA in theblood of the subject.
 41. The method of claim 40, wherein the subjecthas a disease characterized by elevated level of cfDNA in the blood. 42.The method of claim 40, wherein the subject has a disease selected froma neurodegenerative disease, a cancer, a chemotherapy-related toxicity,an irradiation induced toxicity, an organ failure, an organ injury, anorgan infarct, ischemia, an acute vascular event, a stroke,graft-versus-host-disease (GVHD), graft rejection, sepsis, systemicinflammatory response syndrome (SIRS), multiple organ dysfunctionsyndrome (MODS), a traumatic injury, aging, diabetes, atherosclerosis,an autoimmune disorder, eclampsia, infertility, a pregnancy-associatedcomplication, a coagulation disorder, and an infection.
 43. A method oftreating a disease in a subject in need thereof, the method comprising:(a) performing an apheresis procedure comprising diverting blood orplasma from the subject into the apheresis device of claim 1 to produceblood or plasma with reduced levels of the cfDNA; and (b) returning theblood or plasma with reduced levels of the cfDNA to the subject, whereinthe apheresis procedure reduces the level of nucleosome-bound cfDNA,exosome-bound cfDNA, and unbound cfDNA in the blood of the subject.44.-45. (canceled)
 46. The method of claim 40, further comprisingmonitoring the level of cfDNA in the blood of the subject.
 47. Themethod of claim 40, comprising continuing or repeating the apheresisprocedure until the level of cfDNA in the blood of the subject isreduced by at least 25%.
 48. The method of claim 47, comprisingcontinuing or repeating the apheresis procedure until the level of cfDNAis reduced by at least 50%.
 49. The method of claim 48, comprisingcontinuing or repeating the apheresis procedure until the level of cfDNAis reduced by at least 75%. 50.-52. (canceled)
 53. The method of claim40, wherein the unbound cfDNA comprises dsDNA, ssDNA andoligonucleotides.
 54. (canceled)
 55. The device of claim 26, wherein theaffinity matrix comprises a DNA binding protein.
 56. The device of claim55, wherein the DNA binding protein comprises a histone.